Modified vitamin K-dependent polypeptides

ABSTRACT

The invention provides vitamin K-dependent polypeptides with enhanced membrane binding affinity. These polypeptides can be used to modulate clot formation in mammals. Methods of modulating clot formation in mammals are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 09/302,239,filed on Apr. 29, 1999, which is a continuation-in-part of U.S. Ser. No.08/955,636, filed on Oct. 23, 1997, now U.S. Pat. No. 6,017,882.

STATEMENT AS TO FEDERALLY-SPONSORED RESEARCH

Funding for work described herein was provided by the federalgovernment, which has certain rights in the invention.

BACKGROUND OF THE INVENTION

Vitamin K-dependent proteins contain 9 to 13 gamma-carboxyglutamic acidresidues (Gla) in their amino terminal 45 residues. The Gla residues areproduced by enzymes in the liver that utilize vitamin K to carboxylatethe side chains of glutamic acid residues in protein precursors. VitaminK-dependent proteins are involved in a number of biological processes,of which the most well described is blood coagulation (reviewed inFurie, B. and Furie, B. C., 1988, Cell, 53:505-518). Vitamin K-dependentproteins include protein Z, protein S, prothrombin, factor X, factor IX,protein C, factor VII and Gas6. The latter protein functions in cellgrowth regulation. Matsubara et al., 1996, Dev. Biol., 180:499-510. TheGla residues are needed for proper calcium binding and membraneinteraction by these proteins. The membrane contact site of factor X isthought to reside within amino acid residues 1-37. Evans and Nelsestuen,1996, Protein Sci., 5:suppl. 1, 163 Abs. Although the Gla-containingregions of the plasma proteins show a high degree of sequence homology,they have at least a 1000-fold range in membrane affinity. McDonald, J.F. et al., 1997, Biochemistry, 36:5120-5137.

Factor VII functions in the initial stage of blood clotting and may be akey element in forming blood clots. The inactive precursor, or zymogen,has low enzyme activity that is greatly increased by proteolyticcleavage at the R152I153 bond to form factor VIIa. This activation canbe catalyzed by factor Xa as well as by VIIa-tissue factor, an integralmembrane protein found in a number of cell types. Fiore, M. M., et al.,1994, J. Biol. Chem., 269:143-149. Activation by VIIa-tissue factor isreferred to as autoactivation. It is implicated in both the activation(formation of factor VIIa from factor VII) and the subsequent activityof factor VIIa. The most important pathway for activation in vivo is notknown. Factor VIIa can activate blood-clotting factors IX and X.

Tissue factor is expressed at high levels on the surface of some tumorcells. A role for tissue factor, and for factor VIIa, in tumordevelopment and invasion of tissues is possible. Vrana, J. A. et al.,Cancer Res., 56:5063-5070. Cell expression and action of tissue factoris also a major factor in toxic response to endotoxic shock. Dackiw, A.A. et al., 1996, Arch. Sure., 131:1273-1278.

Protein C is activated by thrombin in the presence of thrombomodulin, anintegral membrane protein of endothelial cells. Esmon, N. L. et al.,1982, J. Biol. Chem., 257:859-864. Activated protein C (APC) degradesfactors Va and VIIIa in combination with its cofactor, protein S.Resistance to APC is the most common form of inherited thrombosisdisease. Dahlback, B., 1995, Blood, 85:607-614. Vitamin K inhibitors arecommonly administered as a prophylaxis for thrombosis disease.

Vitamin K-dependent proteins are used to treat certain types ofhemophilia. Hemophilia A is characterized by the absence of activefactor VIII, factor VIIIa, or the presence of inhibitors to factor VIII.Hemophilia B is characterized by the absence of active factor IX, factorIXa. Factor VII deficiency, although rare, responds well to factor VIIadministration. Bauer, K. A., 1996, Haemostasis, 26:155-158, suppl. 1.Factor VIII replacement therapy is limited due to development ofhigh-titer inhibitory factor VIII antibodies in some patients.Alternatively, factor VIIa can be used in the treatment of hemophilia Aand B. Factor IXa and factor VIIIa activate factor X. Factor VIIaeliminates the need for factors IX and VIII by activating factor Xdirectly, and can overcome the problems of factor IX and VIIIdeficiencies with few immunological consequences. Hedner et al., 1993,Transfus. Medi. Rev., 7:78-83; Nicolaisen, E. M. et al., 1996, Thromb.Haemost., 76:200-204. Effective levels of factor VIIa administration areoften high (45 to 90 μg/kg of body weight) and administration may needto be repeated every few hours. Shulmav, S. et al., 1996, Thromb.Haemost., 75:432-436.

A soluble form of tissue factor (soluble tissue factor or sTF) that doesnot contain the membrane contact region has been found to be efficaciousin treatment of hemophilia when co-administered with factor VIIa. See,for example, U.S. Pat. No. 5,504,064. In dogs, sTF was shown to reducethe amount of factor VIIa needed to treat hemophilia. Membraneassociation by sTF-VIIa is entirely dependent on the membrane contactsite of factor VII. This contrasts to normal tissue-factor VIIa complex,which is bound to the membrane through both tissue factor and VII (a).

SUMMARY OF THE INVENTION

It has been discovered that modifications within the γ-carboxyglutamicacid (GLA) domain of vitamin K-dependent polypeptides enhance theirmembrane binding affinities. Vitamin K-dependent polypeptides modifiedin such a manner have enhanced activity and may be used asanti-coagulants, pro-coagulants, or for other functions that utilizevitamin K-dependent proteins. For example, an improved factor VIImolecule may provide several benefits by lowering the dosage of VIIaneeded, reducing the relative frequency of administration and/or byproviding qualitative changes that allow more effective treatment ofdeficiency states.

The invention features vitamin K-dependent polypeptides that include amodified GLA domain that enhances membrane-binding affinity of thepolypeptide relative to a corresponding native vitamin K-dependentpolypeptide. In some embodiments, activity of the vitamin K-dependentpolypeptide also is enhanced. The modified GLA domain is from aboutamino acid 1 to about amino acid 45 and includes at least one amino acidsubstitution. For example, the at least one amino acid substitution canbe at amino acids 2, 5, 9 11, 12, 29, 33, 34, 35, or 36, andcombinations thereof. In particular, the at least one substitution canbe at amino acids 11, 12, 29, 33 or 34, amino acids 2, 5, or 9, aminoacids 11 or 12, amino acids 29 or 33, or amino acids 34, 35 or 36. Themodified GLA domain may include an amino acid sequence, which, in thecalcium saturated state, forms a tertiary structure having a cationiccore with a halo of electronegative charge.

The vitamin K-dependent polypeptide may be, for example, protein C,activated protein C, factor IX, factor IXa or active-site modifiedfactor IXa, factor VII, factor VIIa or active site modified factor VIIa,protein S, protein Z, or factor Xa or active-site modified Xa. Themodified GLA domain of protein C or activated protein C may includesubstitution of a glycine residue at amino acid 12. Furthersubstitutions in the GLA domain of protein C or activated protein C caninclude a glutamic acid residue at amino acid 33 and an aspartic acidresidue at amino acid 34, a glutamine or glutamic acid residue at aminoacid 11, a phenylalanine residue at amino acid 29, an aspartic orglutamic acid residue at amino acid 35, or a glutamic acid residue atamino acid 36. The modified GLA domain of factor VII, factor VIIa, andactive site modified factor VIIa may include at least one amino acidsubstitution at amino acids 11 and 33. For example, a glutamine residueat amino acid 11 and a glutamic acid residue at amino acid 33 may besubstituted. The modified GLA domain also can include an aspartic acidor glutamic acid residue at amino acid 35.

The modified GLA domain of protein S can include a substitution of anisoleucine, leucine, valine, or phenylalanine residue at amino acid 9.Further substitutions can include an aspartic acid or glutamic acidresidue at amino acids 34 or 35. The modified GLA domain also caninclude a phenylalanine residue at amino acid 5, and further can includean amino acid substitution in the thrombin-sensitive loop, such as atamino acids 49, 60, or 70. The modified GLA domain of active-sitemodified Factor IXa can include a phenylalanine residue at amino acids29 or 34, a phenylalanine, leucine, or isoleucine residue at amino acid5, or an aspartic acid or glutamic acid residue at amino acids 34 or 35,and combination thereof.

The modified GLA domain of active-site modified Factor Xa can include aglutamine at amino acid 11 or an aspartic acid or glutamic acid residueat amino acid 35. The modified GLA domain of protein Z can include anasparagine or glutamine residue at amino acid 2 or an aspartic acid orglutamic acid residue at amino acids 34, 35, or 36.

The modified GLA domain of vitamin K-dependent polypeptides further caninclude an inactivated cleavage site. For example, factor VII caninclude an inactivated cleavage site, such as a substitution of analanine residue at amino acid 152.

In another aspect, the invention features a vitamin K-dependentpolypeptide that includes a modified GLA domain that enhances membranebinding affinity and activity of the polypeptide. The modified GLAdomain of such a polypeptide includes at least one amino acid insertionat amino acid 4. The polypeptide can be factor VII or VIIa, protein C oractivated protein C, factor X or Xa, or protein S. For example, thepolypeptide can be factor VII or IIa, and can include the insertion of atyrosine or glycine residue.

The invention also features a mammalian host cell that includes avitamin K-dependent polypeptide. The polypeptide includes a modified GLAdomain that enhances membrane-binding affinity of the polypeptiderelative to a corresponding native vitamin K-dependent polypeptide. Themodified GLA domain includes at least one amino acid substitution asdescribed above. The vitamin K-dependent polypeptide may be, forexample, factor VII or factor VIIa.

The invention also relates to a pharmaceutical composition that includesa pharmaceutically acceptable carrier and an amount of a vitaminK-dependent polypeptide effective to inhibit clot formation in a mammal.The vitamin K-dependent polypeptide includes a modified GLA domain thatenhances membrane-binding affinity of the polypeptide relative to acorresponding native vitamin K-dependent polypeptide. In someembodiments, activity of the polypeptide also is enhanced. The modifiedGLA domain includes at least one amino acid substitution. The vitaminK-dependent polypeptide may be, for example, protein C, activatedprotein C or active site modified factor VIIa, protein S, or active sitemodified factor IXa. The comparison can include an anticoagulant agent(e.g. aspirin).

The invention also features a pharmaceutical composition that includes apharmaceutically acceptable carrier and an amount of a vitaminK-dependent polypeptide effective to increase clot formation in amammal. The vitamin K-dependent polypeptide includes a modified GLAdomain that enhances membrane-binding affinity of the polypeptiderelative to a corresponding native vitamin K-dependent polypeptide. Themodified GLA domain includes at least one amino acid substitution. Thevitamin K-dependent polypeptide may be, for example, factor VII, factorVIIa, factor IX or factor IXa. The pharmaceutical composition may alsoinclude soluble tissue factor.

A method of decreasing clot formation in a mammal is also described. Themethod includes administering an amount of a vitamin K-dependentpolypeptide effective to decrease clot formation in the mammal. Thevitamin K-dependent polypeptide includes a modified GLA domain thatenhances membrane-binding affinity of the polypeptide relative to acorresponding native vitamin K-dependent polypeptide. In someembodiments, activity of the polypeptide also is enhanced. The modifiedGLA domain includes at least one amino acid substitution. The vitaminK-dependent polypeptide may be, for example, protein C, activatedprotein C or active site modified factor VIIa or factor IXa, or proteinS.

The invention also features a method of increasing clot formation in amammal. The method includes administering an amount of a vitaminK-dependent polypeptide effective to increase clot formation in themammal. The vitamin K-dependent polypeptide includes a modified GLAdomain that enhances membrane-binding affinity of the polypeptiderelative to a corresponding native vitamin K-dependent polypeptide. Themodified GLA domain includes at least one amino acid substitution. Thevitamin K-dependent polypeptide may be, for example, factor VII, factorVIIa, factor IX or factor IXa.

In another aspect, the invention features a method for identifying avitamin K-dependent polypeptide having enhanced membrane bindingaffinity and activity. The method includes modifying the GLA domain ofthe polypeptide, wherein modifying includes substituting at least oneamino acid in the GLA domain; monitoring membrane binding affinity andactivity of the polypeptide having the modified GLA domain; andidentifying the modified vitamin K-dependent polypeptide as havingenhanced membrane binding affinity and activity if membrane bindingaffinity activity of the modified polypeptide is enhanced relative to acorresponding native vitamin K-dependent polypeptide. Suitablesubstitutions are described above. The polypeptide can increase clotformation or inhibit clot formation.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the binding, with standard deviations, of wild type VIIa(open circles), VIIQ11E33 (filled circles), and bovine factor X (filledtriangles) to membranes.

FIG. 2 depicts the autoactivation of VIIQ11E33. The dashed line showsactivity in the absence of phospholipid.

FIG. 3 depicts the activation of factor X by factor VIIa. Results forwild type factor VIIa (open circles) and VIIaQ11E33 (filled circles) aregiven for a concentration of 0.06 nM.

FIG. 4 depicts the coagulation of human plasma by VIIa and VIIaQ11E33with soluble tissue factor.

FIG. 5 depicts the coagulation of plasma by factor VII zymogens andnormal tissue factor.

FIG. 6 depicts the inhibition of clot formation by active site modifiedfactor VIIaQ11E33 (DEGR-VIIaQ11E33).

FIG. 7 depicts the circulatory time of factor VIIQ11E33 in rats.

FIG. 8 depicts the membrane interaction by normal and modified proteins.Panel A shows the interaction of wild type bovine protein C (opencircles) and bovine protein C-H11 (filled circles) with vesicles. PanelB shows the interaction of wild type human protein C (open circles) andhuman protein C-P11 (filled circles) with membranes. In both cases, thedashed line indicates the result if all of the added protein were boundto the membrane.

FIG. 9 depicts the influence of activated protein C on clotting times.In panel A, the average and standard deviation for three determinationsof clotting times for bovine plasma are shown for wild type bovine APC(open circles) and for bAPC-H11 (filled circles). In panel B, theaverage and standard deviation of three replicates of human plasmacoagulation for the wild type human (open circles) and human APC-P11(filled circles) is shown.

FIG. 10 depicts the inactivation of factor Va by bovine and human APC.Panel A depicts the inactivation of factor Va by wild type bovine APC(open circles) and bovine APC-H11 (filled circles). Panel B depicts theinactivation of human factor Va in protein S-deficient plasma by eitherwild type human APC (open circles) and human APC-H11 (filled circles).

FIG. 11 depicts the electrostatic distribution of protein Z. Verticallines denote electropositive regions and horizontal lines denoteelectronegative regions

FIG. 12 depicts the membrane binding and activity of various protein Cs.Panel A shows membrane binding by wild type protein C (open circles),the P11H mutant of protein C (filled squares), Q33E, N34D mutant (filledcircles) and bovine prothrombin (open squares). Panel B shows inhibitionof blood coagulation by these mutants. Panel C shows the inactivation offactor Va.

FIG. 13 compares membrane binding and activity of human protein Cmutants. Panel A compares the membrane binding of wild-type (opencircles), E33 (open triangles) and E33D34 (filled circles). Panel Bcompares the coagulation times using wild-type (open triangles), E33(open circles) and E33D34 (filled circles).

FIG. 14 compares membrane binding (Panel A) and coagulation inhibition(Panel B) with wild-type (open squares), H11 (filled circles), E33D34(open triangles) and the triple H11E33D34 mutant (open circles) ofbovine protein C.

FIG. 15 depicts the membrane interaction properties of different vitaminK-dependent proteins. Panel A compares membrane interaction of human(filled circles) and bovine (open circles) factor X. Panel B showsmembrane interaction by normal bovine prothrombin fragment 1 (opencircles), fragment 1 modified with TNBS in the absence of calcium(filled circles) and fragment 1 modified with TNBS in the presence of 25mM calcium (filled squares). Panel C shows the rate of protein Z bindingto vesicles at pH9 (filled circles) and 7.5 (open circles).

FIGS. 16A-16C are clot signature analyzer reports of collagen inducedthrombus formation (CITF) for normal blood (A), for blood containing 30nM wild type APC (B), and blood containing 6 nM Q11G12E33D34 APC(C).

DETAILED DESCRIPTION

In one aspect, the invention features a vitamin K-dependent polypeptideincluding a modified GLA domain with enhanced membrane binding affinityrelative to a corresponding native vitamin K-dependent polypeptide.Activity of the vitamin K-dependent polypeptide also can be enhanced.Enhanced activity depends on the nature of the vitamin K-dependentpolypeptide. For example, enhanced activity of a polypeptide withanticoagulant activity refers to an increase in the clotting time incomparison to a corresponding native polypeptide. Vitamin K-dependentpolypeptides are a group of proteins that utilize vitamin K in theirbiosynthetic pathway to carboxylate the side chains of glutamic acidresidues in protein precursors. The GLA domain contains 9-13γ-carboxyglutamic acid residues in the N-terminal region of thepolypeptide, typically from amino acid 1 to about amino acid 45. ProteinZ, protein S, factor X, factor II (prothrombin), factor IX, protein C,factor VII and Gas6 are examples of vitamin K-dependent polypeptides.Amino acid positions of the polypeptides discussed herein are numberedaccording to factor IX. Protein S, protein C, factor X, factor VII andhuman prothrombin all have one less amino acid (position 4) and must beadjusted accordingly. For example, actual position 10 of bovine proteinC is a proline, but is numbered herein as amino acid 11 for ease ofcomparison throughout. As used herein, the term “polypeptide” is anychain of amino acids, regardless of length or post-translationalmodification. Amino acids have been designated herein by standard threeletter and one-letter abbreviations.

Modifications of the GLA domain include at least one amino acidsubstitution (e.g, one to 10 substitutions). The substitutions may beconservative or non-conservative. Conservative amino acid substitutionsreplace an amino acid with an amino acid of the same class, whereasnon-conservative amino acid substitutions replace an amino acid with anamino acid of a different class. Non-conservative substitutions mayresult in a substantial change in the hydrophobicity of the polypeptideor in the bulk of a residue side chain. In addition, non-conservativesubstitutions may make a substantial change in the charge of thepolypeptide, such as reducing electropositive charges or introducingelectronegative charges. Examples of non-conservative substitutionsinclude a basic amino acid for a non-polar amino acid, or a polar aminoacid for an acidic amino acid. The amino acid substitution may be atamino acid 2, 5, 9, 11, 12, 29, 33, 34, 35, or 36, and combinationsthereof. The modified GLA domain may include an amino acid sequence,which, in the calcium-saturated state, contributes to formation of atertiary structure having a cationic core with a halo of electronegativecharge. The highest affinity proteins show an electronegative chargeextending to amino acids 35 and 36. Without being bound by a particulartheory, enhanced membrane affinity may result from a particularelectrostatic pattern consisting of an electropositive core completelysurrounded by an electronegative surface.

In addition, modifications of the GLA domain can include an insertion ofa residue at position four of a vitamin K-dependent polypeptide.Suitable polypeptides lack an amino acid at this position (Protein S,protein C, factor X, factor VII, and human prothrombin), based onsequence alignments of vitamin K-dependent polypeptides.

Many vitamin K-dependent polypeptides are substrates for membrane-boundenzymes. Since no vitamin K-dependent polypeptides display the maximumpotential membrane-binding affinity of a GLA domain, all must containamino acids whose purpose is to reduce binding affinity. Consequently,many vitamin K-dependent polypeptides contain amino acids that arenon-optimal from the standpoint of maximum membrane-binding affinity.These residues effectively disrupt the binding site to provide a morerapid turnover for an enzymatic reaction.

Lowered membrane affinity may serve several purposes. High affinity isaccompanied by slow exchange, which may limit reaction rates. Forexample, when the prothrombinase enzyme is assembled on membranes withhigh affinity for substrate, protein exchange from the membrane, ratherthan enzyme catalysis, is the limiting step. Lu, Y. and Nelsestuen, G.L., 1996, Biochemistry, 35:8201-8209. Alternatively, adjustment ofmembrane affinity by substitution with non-optimum amino acids maybalance the competing processes of procoagulation (factor X, IX, VII,and prothrombin) and anticoagulation (protein C, S). Although membraneaffinities of native proteins may be optimal for normal states,enhancement of membrane affinity can produce proteins that are usefulfor in vitro study as well as improved therapeutics for regulating bloodclotting in pathological conditions in vivo.

Modified vitamin K-dependent polypeptides having enhanced membranebinding affinity and activity can be identified by monitoring membranebinding affinity and activity of the vitamin K-dependent polypeptide.For example, membrane binding can be assessed using large or smallunilamellar vesicles prepared from various ratios of phosphotidylserineand phosphatidylcholine. Light scattering intensity at 90° is measuredfor the vesicles alone or after addition of a polypeptide. Activity ofthe modified vitamin K-dependent polypeptides can be measured, forexample, by measuring coagulation time. Factor VIIa clotting activitycan be assessed using saturating amounts of Factor VIIa, reconstitutedtissue factor, and membrane. After addition of factor VII-deficientplasma, clotting time is measured. In vivo conditions can be mimicked byaddition of a competing protein such as active site modified factorVIIa. Activity of activated protein C can be assessed using, forexample, a clot signature analyzer (Xylon Company, Scarsdale, N.Y.) todetermine the average collagen-induced thrombus formation time.

Various examples of GLA domain modified vitamin K-dependent polypeptidesare described below.

The vitamin K-dependent polypeptide may be protein C or activatedprotein C (APC). Amino acid sequences of the wild-type human (hC, SEQ IDNO:1) and bovine (bC, SEQ ID NO:2) protein C GLA domains are shown inTable 1. X is a Gla or Glu residue. In general, a protein with neutral(e.g., Q) or anionic residues (e.g., D, E) at positions 11, 33, 34, 35,and 36 will have higher membrane affinity. TABLE 1 hC:ANS-FLXXLRH₁₁SSLXRXCIXX₂₁ICDFXXAKXI₃₁FQNVDDTLAF₄₁ WSKH bC:ANS-FLXXLRP₁₁GNVXRXCSXX₂₁VCXFXXARXI₃₁FQNTXDTMAF₄₁ WSFY

The GLA domain of protein C or APC can contain a substitution at aminoacids 11, 12, 29, 33, 34, 35, or 36, and combinations thereof. Forexample, the modified GLA domain may include a substitution at aminoacid 12 of a glycine residue for serine, and further may include aglutamic acid residue at amino acid 33 and an aspartic acid or glutamicacid residue at amino acid 34. The glutamic acid at position 33 may befurther modified to γ-carboxyglutamic acid in vivo. For optimumactivity, the modified GLA domain may include an additional substitutionat amino acid 11. For example, a glutamine residue may be substituted atamino acid 11 or alternatively, a glutamic acid or an aspartic acidresidue may be substituted. A histidine residue also may be substitutedat amino acid 11 in bovine protein C. Replacement of amino acid 29 byphenylalanine, the amino acid found in prothrombin, is another usefulmodification. Glutamic acid or aspartic acid residues also can besubstituted at amino acid 35. Modified protein C with enhanced membranebinding affinity may be used in place of, or in combination with, otherinjectable anticoagulants such as heparin. Heparin is typically used inmost types of surgery, but suffers from a low efficacy/toxicity ratio.In addition, modified protein C with enhanced membrane affinity may beused in place of, or in combination with, oral anticoagulants, includingaspirin and anticoagulants in the coumarin family, such as warfarin.

These modifications also can be made with active site modified APC. Theactive site of APC may be inactivated chemically, for example byN-dansyl-glutamyl glycylarginylchloromethylketone (DEGR),phenylalanyl-phenylalanyl arginylchloromethylketone (FFR), or bysite-directed mutagenesis of the active site. Sorensen, B. B. et al.,1997, J. Biol. Chem., 272:11863-11868. Active site-modified APCfunctions as an inhibitor of the prothrombinase complex. Enhancedmembrane affinity of active site modified APC may result in a moretherapeutically effective polypeptide.

The vitamin K-dependent polypeptide may be factor VII or the active formof factor VII, factor VIIa. Native or naturally occurring factor VIIpolypeptide has low affinity for membranes. Amino acid sequences of thewild-type human (hVII, SEQ ID NO: 3) and bovine (bVII, SEQ ID NO: 4)factor VII GLA domains are shown in Table 2. TABLE 2 hVII:ANA-FLXXLRP₁₁GSLXRXCKXX₂₁QCSFXXARXI₃₁FKDAXRTKLF₄₁ WISY bVII:ANG-FLXXLRP₁₁GSLXRXCRXX₂₁LCSFXXAHXI₃₁FRNXXRTRQF₄₁ WVSY

The GLA domain of factor VII or VIIa can contain a substitution, forexample at amino acid 11, 29, 33, 34, or 35, and combinations thereof.The modified GLA domain of factor VII or factor VIIa may include, forexample, a glutamic acid, a glutamine, an asparagine, or an asparticacid residue at amino acid 11, a phenylalanine or a glutamic acidresidue at amino acid 29, or an aspartic acid or a glutamic acid residueat amino acids 33 or 35. Other neutral residues also may be substitutedat these positions. The modified GLA domain can include combinations ofsuch substitutions at amino acid residues 11 and 29, at residues 11 and33, at residues 11 and 35, at residues 11, 33, and 35, at residues 11,29, and 33, at residues 11, 29, 33, and 35, at residues 29 and 33, atresidues 29 and 35, or at residues 29, 33, and 35. For example, the GLAdomain of factor VII or factor VIIa may include a glutamine residue atamino acid 11 and a glutamic acid residue at amino acid 33, or aglutamine residue at amino acid 11 and a phenylalanine residue at aminoacid 29. The modified GLA domain also may include a substitution atamino acid 34 in combination with one or more substitutions at positions11, 29, 33, and 35. For example, a phenylalanine residue can besubstituted at amino acid 34. In addition, the modified GLA domain caninclude an insertion at position 4 alone (e.g, a tyrosine or glycineresidue) or in combination with substitutions described above.

Factor VII or VIIa modified in these manners has a much higher affinityfor membranes than the native or wild type polypeptide. It also has amuch higher activity in autoactivation, in factor Xa generation and inseveral blood clotting assays. Activity is particularly enhanced atmarginal coagulation conditions, such as low levels of tissue factorand/or phospholipid. For example, modified factor VII is about 4 timesas effective as native VIIa at optimum thromboplastin levels, but isabout 20-fold as effective at 1% of optimum thromboplastin levels.Marginal pro-coagulation signals are probably most predominant in vivo.Presently available clotting assays that use optimum levels ofthromboplastin cannot detect clotting time differences between normalplasma and those from hemophilia patients. Clotting differences betweensuch samples are only detected when non-optimal levels of thromboplastinor dilute thromboplastin are used in clotting assays.

Another example of a vitamin K-dependent polypeptide is active sitemodified Factor VIIa. The active site of factor VIIa may be modifiedchemically, for example by DEGR, FFR, or by site-directed mutagenesis ofthe active site. DEGR-modified factor VII is an effective inhibitor ofcoagulation by several routes of administration. Arnljots, B. et al.,1997, J. Vasc. Surg., 25:341-346. Modifications of the GLA domain maymake active site modified Factor VIIa more efficacious. Suitablesubstitutions or insertions are described above.

The vitamin K-dependent polypeptide may also be Factor IX or the activeform of Factor IX, Factor IXa. As with active site-modified factor VIIa,active site modified IXa may be an inhibitor of coagulation. Active sitemodified factor IXa can bind its cofactor, Factor VIII, but will notform blood clots. Active site modified Factor IXa (wild-type) preventscoagulation without increase of bleeding in an animal model of stroke.See, for example, Choudhri et al., J. Exp. Med., 1999, 190:91-99.

Amino acid sequences of the wild-type human (hIX, SEQ ID NO:5) andbovine (bIX, SEQ ID NO:6) factor IX GLA domains are shown in Table 3.For example, a valine, leucine, phenylalanine, or isoleucine residue maybe substituted at amino acid 5, an aspartic acid or glutamic acidresidue may be substituted at amino acid 11, a phenylalanine residue atamino acid 29, or an aspartic acid or glutamic acid residue at aminoacids 34 or 35, and combinations thereof. TABLE 3 hIX:YNSGKLXXFVQ₁₁GNLXRXCMXX₂₁KCSFXXARXV₃₁FXNTXRTTXF₄₁ WKQY bIX:YNSGKLXXFVQ₁₁GNLXRXCMXX₂₁KCSFXXARXV₃₁FXNTXKRTTXF₄₁ WKQY

A further example of a vitamin K-dependent polypeptide is protein S. Theamino acid sequence of human protein S (hPS, SEQ ID NO:19) is shown inTable 4. The modified GLA domain of Protein S can have, for example, asubstitution at amino acids 5, 9, 34, or 35, and combinations thereof.For example, a phenylalanine can be substituted at amino acid 5, anisoleucine, leucine, valine, or phenylalanine residue at amino-acid 9,or an aspartic acid or glutamic acid residue at amino acids 34 or 35. Inaddition to the at least one substitution in the GLA domain, protein Sfurther can include a substitution in the thrombin sensitive loop. Inparticular, residues 49, 60, or 70 of the thrombin sensitive loop, whicheach are arginine residues, can be replaced with, for example, alanineresidues. TABLE 4 hPS: ANS-LLXXTKQ₁₁GNLXRXCIXX₂₁LCNKXXARXV₃₁FXNDPXTDYF₄₁YPKY

The vitamin K-dependent polypeptides of the invention also can includean inactivated cleavage site such that the polypeptides are notconverted to an active form. For example, Factor VII containing aninactivated cleavage site would not be converted to Factor VIIa, butwould still be able to bind tissue factor. In general, an arginineresidue is found at the cleavage site of vitamin K-dependentpolypeptides. Any residue can be substituted for the arginine at thisposition to inactivate the cleavage site. In particular, an alanineresidue could be substituted at amino acid 152 of Factor VII. VitaminK-dependent polypeptides of the invention that further contain aninactivated cleavage site act as inhibitors.

In another aspect, the invention features a mammalian host cellincluding a vitamin K-dependent polypeptide having a modified GLA domainthat enhances membrane-binding affinity of the polypeptide relative to acorresponding native vitamin K-dependent polypeptide. Activity of thevitamin K-dependent polypeptides can be enhanced in some embodiments.Suitable vitamin K-dependent polypeptides and modifications of the GLAdomain are discussed above. The mammalian host cell may include, forexample, modified factor VII or modified factor VIIa. The GLA domain ofmodified factor VII or modified factor VIIa may contain an amino acidsubstitution at amino acid 11 and at amino acid 33. Preferably, theamino acid substitution includes a glutamine residue at amino acid 11and a glutamic acid residue at amino acid 33 of factor VII or VIIa.Suitable mammalian host cells are able to modify vitamin K-dependentpolypeptide glutamate residues to γ-carboxyglutamate. Mammalian cellsderived from kidney and liver are especially useful as host cells.

Nucleic Acids Encoding Modified Vitamin K-Dependent Polypeptides

Isolated nucleic acid molecules encoding modified vitamin K-dependentpolypeptides of the invention can be produced by standard techniques. Asused herein, “isolated” refers to a sequence corresponding to part orall of a gene encoding a modified vitamin K-dependent polypeptide, butfree of sequences that normally flank one or both sides of the wild-typegene in a mammalian genome. An isolated polynucleotide can be, forexample, a recombinant DNA molecule, provided one of the nucleic acidsequences normally found immediately flanking that recombinant DNAmolecule in a naturally-occurring genome is removed or absent. Thus,isolated polynucleotides include, without limitation, a DNA moleculethat exists as a separate molecule (e.g., a cDNA or genomic DNA fragmentproduced by PCR or restriction endonuclease treatment) independent ofother sequences as well as recombinant DNA that is incorporated into avector, an autonomously replicating plasmid, a virus (e.g., aretrovirus, adenovirus, or herpes virus), or into the genomic DNA of aprokaryote or eukaryote. In addition, an isolated polynucleotide caninclude a recombinant DNA molecule that is part of a hybrid or fusionpolynucleotide.

It will be apparent to those of skill in the art that a polynucleotideexisting among hundreds to millions of other polynucleotides within, forexample, cDNA or genomic libraries, or gel slices containing a genomicDNA restriction digest, is not to be considered an isolatedpolynucleotide.

Isolated nucleic acid molecules are at least about 14 nucleotides inlength. For example, the nucleic acid molecule can be about 14 to 20,20-50, 50-100, or greater than 150 nucleotides in length. In someembodiments, the isolated nucleic acid molecules encode a full-lengthmodified vitamin K-dependent polypeptide. Nucleic acid molecules can beDNA or RNA, linear or circular, and in sense or antisense orientation.

Specific point changes can be introduced into the nucleic acid sequenceencoding wild-type vitamin K-dependent polypeptides by, for example,oligonucleotide-directed mutagenesis. In this method, a desired changeis incorporated into an oligonucleotide, which then is hybridized to thewild-type nucleic acid. The oligonucleotide is extended with a DNApolymerase, creating a heteroduplex that contains a mismatch at theintroduced point change, and a single-stranded nick at the 5′ end, whichis sealed by a DNA ligase. The mismatch is repaired upon transformationof E. coli or other appropriate organism, and the gene encoding themodified vitamin K-dependent polypeptide can be re-isolated from E. colior other appropriate organism. Kits for introducing site-directedmutations can be purchased commercially. For example, Muta-Gene7in-vitro mutagenesis kits can be purchased from Bio-Rad Laboratories,Inc. (Hercules, Calif.).

Polymerase chain reaction (PCR) techniques also can be used to introducemutations. See, for example, Vallette et al., Nucleic Acids Res., 1989,17(2):723-733. PCR refers to a procedure or technique in which targetnucleic acids are amplified. Sequence information from the ends of theregion of interest or beyond typically is employed to designoligonucleotide primers that are identical in sequence to oppositestrands of the template to be amplified, whereas for introduction ofmutations, oligonucleotides that incorporate the desired change are usedto amplify the nucleic acid sequence of interest. PCR can be used toamplify specific sequences from DNA as well as RNA, including sequencesfrom total genomic DNA or total cellular RNA. Primers are typically 14to 40 nucleotides in length, but can range from 10 nucleotides tohundreds of nucleotides in length. General PCR techniques are described,for example in PCR Primer: A Laboratory Manual, Ed. by Dieffenbach, C.and Dveksler, G., Cold Spring Harbor Laboratory Press, 1995.

Nucleic acids encoding modified vitamin K-dependent polypeptides alsocan be produced by chemical synthesis, either as a single nucleic acidmolecule or as a series of oligonucleotides. For example, one or morepairs of long oligonucleotides (e.g., >100 nucleotides) can besynthesized that contain the desired sequence, with each pair containinga short segment of complementarity (e.g., about 15 nucleotides) suchthat a duplex is formed when the oligonucleotide pair is annealed. DNApolymerase is used to extend the oligonucleotides, resulting in adouble-stranded nucleic acid molecule per oligonucleotide pair, whichthen can be ligated into a vector.

Production of Modified Vitamin K-Dependent Polypeptides

Modified vitamin K-dependent polypeptides of the invention can beproduced by ligating a nucleic acid sequence encoding the polypeptideinto a nucleic acid construct such as an expression vector, andtransforming a bacterial or eukaryotic host cell with the expressionvector. In general, nucleic acid constructs include a regulatorysequence operably linked to a nucleic acid sequence encoding a vitaminK-dependent polypeptide. Regulatory sequences do not typically encode agene product, but instead affect the expression of the nucleic acidsequence. As used herein, “operably linked” refers to connection of theregulatory sequences to the nucleic acid sequence in such a way as topermit expression of the nucleic acid sequence. Regulatory elements caninclude, for example, promoter sequences, enhancer sequences, responseelements, or inducible elements.

In bacterial systems, a strain of E. coli such as BL-21 can be used.Suitable E. coli vectors include without limitation the pGEX series ofvectors that produce fusion proteins with glutathione S-transferase(GST). Transformed E. coli are typically grown exponentially thenstimulated with isopropylthiogalactopyranoside (IPTG) prior toharvesting. In general, such fusion proteins are soluble and can bepurified easily from lysed cells by adsorption to glutathione-agarosebeads followed by elution in the presence of free glutathione. The pGEXvectors are designed to include thrombin or factor Xa protease cleavagesites such that the cloned target gene product can be released from theGST moiety.

In eukaryotic host cells, a number of viral-based expression systems canbe utilized to express modified vitamin K-dependent polypeptides. Anucleic acid encoding vitamin K-dependent polypeptide can be clonedinto, for example, a baculoviral vector such as pBlueBac (Invitrogen,San Diego, Calif.) and then used to co-transfect insect cells such asSpodoptera frugiperda (Sf9) cells with wild-type DNA from Autographacalifornica multiply enveloped nuclear polyhedrosis virus (AcMNPV).Recombinant viruses producing the modified vitamin K-dependentpolypeptides can be identified by standard methodology. Alternatively, anucleic acid encoding a vitamin K-dependent polypeptide can beintroduced into a SV40, retroviral, or vaccinia based viral vector andused to infect suitable host cells.

Mammalian cell lines that stably express modified vitamin K-dependentpolypeptides can be produced by using expression vectors with theappropriate control elements and a selectable marker. For example, theeukaryotic expression vector pCDNA.3.1+(Invitrogen, San Diego, Calif.)is suitable for expression of modified vitamin K-dependent polypeptidesin, for example, COS cells, HEK293 cells, or baby hamster kidney cells.Following introduction of the expression vector by electroporation, DEAEdextran-, calcium phosphate-, liposome-mediated transfection, or othersuitable method, stable cell lines can be selected. Alternatively,transiently transfected cell lines are used to produce modified vitaminK-dependent polypeptides. Modified vitamin K-dependent polypeptides alsocan be transcribed and translated in vitro using wheat germ extract orrabbit reticulocyte lysate.

Modified vitamin K-dependent polypeptides can be purified fromconditioned cell medium by applying the medium to an immunoaffinitycolumn. For example, an antibody having specific binding affinity forFactor VII can be used to purify modified Factor VII. Alternatively,concanavalin A (Con A) chromatography and anion-exchange chromatography(e.g., DEAE) can be used in conjunction with affinity chromatography topurify factor VII. Calcium dependent or independent monoclonalantibodies that have specific binding affinity for factor VII can beused in the purification of Factor VII.

Modified vitamin K-dependent polypeptides such as modified protein C canbe purified by anion-exchange chromatography, followed by immunoaffinitychromatography using an antibody having specific binding affinity forprotein C.

Modified vitamin K-dependent polypeptides also can be chemicallysynthesized using standard techniques. See, Muir, T. W. and Kent, S. B.,Curr. Opin. Biotechnol., 1993, 4(4):420-427, for a review of proteinsynthesis techniques.

Pharmaceutical Compositions

The invention also features a pharmaceutical composition including apharmaceutically acceptable carrier and an amount of a vitaminK-dependent polypeptide effective to inhibit clot formation in a mammal.The vitamin K-dependent polypeptide includes a modified GLA domain withat least one amino acid substitution or insertion that enhancesmembrane-binding affinity of the polypeptide relative to a correspondingnative vitamin K-dependent polypeptide. In some embodiments, activity ofthe vitamin K-dependent polypeptide also is enhanced. Useful modifiedvitamin K-dependent polypeptides of the pharmaceutical compositions caninclude, without limitation, protein C or APC, active-site modified APC,active-site modified factor VIIa, active-site modified factor IXa,active-site modified factor Xa, or Protein S, as discussed above.Pharmaceutical compositions also can include an anticoagulant agent suchas aspirin, warfarin, or heparin.

The concentration of a vitamin K-dependent polypeptide effective toinhibit clot formation in a mammal may vary, depending on a number offactors, including the preferred dosage of the compound to beadministered, the chemical characteristics of the compounds employed,the formulation of the compound excipients and the route ofadministration. The optimal dosage of a pharmaceutical composition to beadministered may also depend on such variables as the overall healthstatus of the particular patient and the relative biological efficacy ofthe compound selected. These pharmaceutical compositions may be used toregulate coagulation in vivo. For example, the compositions may be usedgenerally for the treatment of thrombosis. Altering only a few aminoacid residues of the polypeptide as described above, generally does notsignificantly affect the antigenicity of the mutant polypeptides.

Vitamin K-dependent polypeptides that include modified GLA domains maybe formulated into pharmaceutical compositions by admixture withpharmaceutically acceptable non-toxic excipients or carriers. Suchcompounds and compositions may be prepared for parenteraladministration, particularly in the form of liquid solutions orsuspensions in aqueous physiological buffer solutions; for oraladministration, particularly in the form of tablets or capsules; or forintranasal administration, particularly in the form of powders, nasaldrops, or aerosols. Compositions for other routes of administration maybe prepared as desired using standard methods.

Formulations for parenteral administration may contain as commonexcipients (pharmaceutically acceptable carriers) sterile water orsaline, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, hydrogenated naphthalenes, and the like. Inparticular, biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxethylene-polyoxypropylenecopolymers are examples of excipients for controlling the release of acompound of the invention in vivo. Other suitable parenteral deliverysystems include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. Formulations forinhalation administration may contain excipients such as lactose, ifdesired. Inhalation formulations may be aqueous solutions containing,for example, polyoxyethylene-9-lauryl ether, glycocholate anddeoxycholate, or they may be oily solutions for administration in theform of nasal drops. If desired, the compounds can be formulated as gelsto be applied intranasally. Formulations for parenteral administrationmay also include glycocholate for buccal administration.

In an alternative embodiment, the invention also features apharmaceutical composition including a pharmaceutically acceptablecarrier and an amount of a vitamin K-dependent polypeptide effective toincrease clot formation in a mammal. The vitamin K-dependent polypeptideincludes a modified GLA domain with at least one amino acid substitutionthat enhances membrane binding affinity of the polypeptide relative to acorresponding native vitamin K-dependent polypeptide. Thesepharmaceutical compositions may be useful for the treatment of clottingdisorders such as hemophilia A, hemophilia B and liver disease.

In this embodiment, useful vitamin K-dependent polypeptides of thepharmaceutical compositions can include, without limitations, Factor VIIor the active form of Factor VII, Factor VIIa. The modified GLA domainof Factor VII or Factor VIIa may include substitutions at amino acid 11and amino acid 33, for example, a glutamine residue at amino acid 11 anda glutamic acid residue at amino acid 33. The pharmaceutical compositionmay further comprise soluble tissue factor. Factor VII is especiallycritical to blood coagulation because of its location at the initiationof the clotting cascade, and its ability to activate two proteins,factors IX and X. Direct activation of factor X by factor VIIa isimportant for possible treatment of the major forms of hemophilia, typesA and B, since the steps involving factors IX and VIII are bypassedentirely. Administration of factor VII to patients has been found to beefficacious for treatment of some forms of hemophilia. Improvement ofthe membrane affinity of factor VII or VIIa by modification of the GLAdomain provides the potential to make the polypeptide more responsive tomany coagulation conditions, to lower the dosages of VII/VIIa needed, toextend the intervals at which factor VII/VIIa must be administered, andto provide additional qualitative changes that result in more effectivetreatment. Overall, improvement of the membrane contact site of factorVII. may increase both its activation rate as well as improve theactivity of factor VIIa on factor X or IX. These steps may have amultiplicative effect on overall blood clotting rates in vivo, resultingin a very potent factor VIIa for superior treatment of several bloodclotting disorders.

Other useful vitamin K-dependent polypeptides for increasing clotformation include Factor IX and Factor IXa, and Factor X and Xa.

In another aspect, methods for decreasing clot formation in a mammal aredescribed. The method includes administering an amount of vitaminK-dependent polypeptide effective to decrease clot formation in themammal. The vitamin K-dependent polypeptide includes a modified GLAdomain that enhances membrane-binding affinity of the polypeptiderelative to a corresponding native vitamin K-dependent polypeptide. Insome embodiments, activity also is enhanced. The modified GLA domainincludes at least one amino acid substitution. Modified protein C orAPC, protein S, or active-site modified factors VIIa, IXa, and Xacontaining substitutions in the GLA domain may be used for this method.The method further can include administering an anticoagulant agent.

In another aspect, the invention also features methods for increasingclot formation in a mammal that includes administering an amount ofvitamin K-dependent polypeptide effective to increase clot formation inthe mammal. The vitamin K-dependent polypeptide includes a modified GLAdomain that enhances membrane-binding affinity of the polypeptiderelative to a corresponding native vitamin K-dependent polypeptide. Themodified GLA domain includes at least one amino acid substitution.Modified factor VII or VIIa and modified factor IX or IXa may be used inthis method.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Factor VII with Enhanced Membrane Affinity andActivity

It has been found that the membrane binding affinity of human bloodclotting factor VII can be increased by site-directed mutagenesis. Theproperties of a P11Q,K33E mutant (referred to herein as Factor VIIQ11E33or mutant factor VII) have been characterized. Membrane affinity wasincreased over wild type protein by about 20-fold. Autoactivation by themutant was increased by at least 100-fold over that of wild type factorVII. The activated form of VIIQ11E33 (referred to as VIIaQ11E33)displayed about 10-fold higher activity toward factor X. The coagulationactivity of VIIaQ11E33 with soluble tissue factor in normal plasma wasabout 10-fold higher than that of wild type VIIa. Coagulation activityof the zymogen, VIIQ11E33, with normal tissue factor (supplied as a1:100 dilution of thromboplastin-HS), was 20-fold higher than wild typeFactor VII. The degree to which activity was enhanced was dependent onconditions, with VIIQ11E33 being especially active under conditions oflow coagulation stimuli.

In general, protein concentrations were determined by the Bradford assayusing bovine serum albumin as the standard. Bradford, M. M., 1976,Analyt. Biochem. 248-254. Molar concentrations were obtained from themolecular weights of 50,000 for factor VII and 55,000 for factor X.Unless indicated, all activity measurements were conducted in standardbuffer (0.05 M Tris, pH7.5, 100 mM NaCl).

Production of Mutant Factor VII: Mutant factor VII was generated fromwild type factor VII cDNA (GenBank Accession number M13232, NIDg182799). Petersen et al., 1990, Biochemistry 29:3451-3457. The P11Qmutation (change of amino acid 11 from a proline residue to a glutamineresidue) and the K33E mutation (change of amino acid 33 from a lysineresidue to a glutamic acid residue) were introduced into the wild typefactor VII cDNA by a polymerase chain reaction strategy essentially asdescribed by Vallette et al., 1989, Nucleic Acids Res. 17:723-733.During this process, a mutation-diagnostic XmaIII restriction enzymesite was eliminated. Four PCR primers were designed to prime synthesisof two mutant fragments of M13232, one from MluI to BglII, positions 221to 301, and the other from BglII to SstII, positions 302 to 787. Theseprimers were used under standard PCR cycling conditions (GENEAMP, PerkinElmer) to prime fragment synthesis using 1 ng of the wild-type factorVII cDNA as template. The resulting fragments were gel purified anddigested with MluI and BglII or BglII and SstII. The two purifiedfragments were then ligated into the factor VIII cDNA in the expressionvector Zem219b from which the corresponding wild-type sequence had beenremoved as a MluI-SstII fragment. Petersen et al., 1990 supra. Themutated fragments were sequenced in their entirety to confirm the P11Qand K33E substitutions, as well as to eliminate the possibility of otherPCR-induced sequence changes.

Transfection, Selection and Purification: Baby hamster kidney (BHK)cells were grown in Dubeccos modified Eagles medium supplemented with10% fetal calf serum and penicillin-streptomycin. Subconfluent cellswere transfected with the factor VII expression plasmid usinglipofectAMINE (Gibco BRL) according to the manufacturersrecommendations. Two days post-transfection, cells were trypsinized anddiluted to selective medium containing 1 μM methotrexate (MTX).Stably-transfected BHK cells were subsequently cultured in serum-freeDubeccos modified Eagles medium supplemented withpenicillin-streptomycin, 5 μg/mL vitamin K₁ and 1 μM MTX, andconditioned medium was collected. The conditioned medium was appliedtwice to an immunoaffinity column composed of a calcium-dependentmonoclonal antibody (CaFVII22) coupled to Affi-Gel 10. Nakagaki et al.,1991, Biochemistry, 30:10819-10824. The final purified Factor VIIQ11E33ran as a single band on SDS polyacrylamide gel electrophoresis, with noevidence of factor VIIa in the preparation. The pure VII(P11Q,K33E)mutant showed 1400-2800 factor VII units/mg.

Activation of Factor VII: Activated Factor VIIaQ11E33 was formed bybovine factor Xa cleavage of VIIQ11E33 (1:100 weight ratio, incubationfor 1 hr at 37° C.). Alternatively, Factor VIIaQ11E33 was obtained byautoactivation (37° C., 20 min) in a mixture containing 7 μM VIIQ11E33,0.7 μM sTF and phospholipid (phosphatidylserine/phosphatidylcholine(PS/PC), 25/75, 0.1 g/g protein).

Wild-type factor VIIa was a homogeneous, recombinant protein (NOVONordisk). Two preparations consisted of a commercial, lyophilizedproduct and non-lyophilized product. The latter protein was furtherpurified on FPLC mono-Q and showed a specific activity of 80,000units/mg, calibrated with a George King NPP standard.

Enhanced membrane interaction by Factor VIIQ11E33: Phospholipidpreparation, assay and measurement of protein-membrane binding wasconducted by the method described by Nelsestuen and Lim, 1977,Biochemistry, 30:10819-10824. Large unilamellar vesicles (LUVs) andsmall unilamellar vesicles (SUVs) were prepared by methods describedpreviously. Hope, M. J., et al., Biochem. Biophys. Acta., 812:55-65;Huang, C., 1969, Biochemistry, 8:344-352. Highly pure phosphatidylserine(bovine brain) and egg phosphatidylcholine (Sigma Chemical Co.) weremixed in chloroform. The solvent was removed by a stream of nitrogengas. The dried phospholipids were suspended in buffer. SUVs were formedby sonication and gel filtration while LUVs were formed by freeze-thawand extrusion. Phospholipid concentrations were determined by organicphosphate assay assuming a phosphorous:phospholipid weight ratio of 25.

SUVS of either PS/PC (25/75) or PS/PC (10/90) were prepared. Protein wasadded to phospholipid at the weight ratios shown in FIG. 1.Protein-membrane binding was assayed by light scattering at 90° by themethod of Nelsestuen and Lim, 1977 supra. Briefly, the light scatteringintensity of phospholipid vesicles alone (II) and after addition ofprotein (I₂) were measured and corrected for background from buffer andunbound protein. The molecular weight ratio of the protein-vesiclecomplex (M₂) to that of the vesicles alone (M1), can be estimated fromthe relationship in equation 1, where δn/δc is the refractive index ofthe respective species.I ₂ /I ₁=(M ₂ /M ₁)²(δn/δc ₂ /δn/δc ₁)²  (eq. 1)If phospholipid and protein concentrations are known, the concentrationof bound [P*PL] and free protein [P] can be estimated. These values,together with the maximum protein binding capacity [P*PL_(max)] of thevesicles (assumed to be 1.0 g/g for all proteins) can be used to obtainthe equilibrium constant for protein-membrane interaction by therelationship in equation 2, where all concentrations are expressed asmolar protein or protein binding sites.K _(D) =[P][P*PL _(max) −P*PL]/[P*PL]  (eq. 2)Binding was assessed at 5 mM calcium and is expressed as the ratio,M2/M1.

FIG. 1 shows the binding of wild type VIIa (open circles) and factorVIIQ11E33 (filled circles) to membranes of either PS/PC=25/75, 25 μg/ml(FIG. 1A) or PS/PC=10/90, 25 μg/ml (FIG. 1B). VIIQ11E33 had much higheraffinity than wild type protein. Binding to PS/PC (25/75) was at thequantitative level so that [Protein_(free)] was essentially zero.Consequently, Kd values could not be estimated from this data. Membranebinding of bovine factor X (filled triangles) is shown in FIG. 1 as areference. Bovine factor X is one of the highest affinity proteins inthis family, giving a Kd for PS/PC (20/80) at 2 mM calcium of 40 nM.McDonald et al., 1997, Biochemistry, 36:5120-5127. The Kd for bovinefactor X, obtained from the result at a protein/phospholipid ratio of0.55 (FIG. 1), was 0.025 μM.

Binding of wild-type and mutant Factor VII to membranes of PS/PC (10/90)was also determined (FIG. 1B). The VIIQ11E33 bound at less than thequantitative level, which allowed a binding constant to be estimatedfrom the relationship in equation 3.Kd=[Protein _(free)][Binding sites_(free)]/[Protein_(bound)]  (eq. 3)[Binding sites_(free)] were estimated from equation 4, assuming amaximum M2/M1 of 1.0 (i.e., [Bindingsites_(total)]=[Phospholipid_(weight conc.)/Protein_(MW)]). This is acommon value observed for several proteins of this family. See McDonaldet al., 1997, supra.[Binding sites_(free)]=[Binding sites_(total)]−[Protein_(bound)]  (eq.4)Using these assumptions and the data at a protein to phospholipid ratioof 0.37, Kd values were 0.7 μM for bovine factor X, 5.5 μM for wild typefactor VII and 0.23 μM for VIIQ11E33. Thus, it was clear that factorVIIQ11E33 was greatly improved in membrane binding affinity over wildtype factor VII and had one of the highest membrane-binding affinitiesamong the vitamin K-dependent proteins.

It also has been observed that the difference between wild-type VIIa andVIIa-Q11E33 varied somewhat with the composition of the phospholipidvesicles that were used. For example, membranes containing PS/PE/PC(20/40/40) produced a 33-fold higher activity for VIIaQ11E33, whilecertain preparations of PS/PC (20/80 to 25/75) showed a 10 to 19 foldhigher activity for VIIaQ11E33.

Enhanced activation of factor VIIQ11E33: The first step in coagulationinvolves the activation of factor VII. Autoactivation of VII wasconducted in a solution containing 100 nM sTF (highly purifiedrecombinant product from Dr. Walter Kisiel, Fiore et al., 1994, J. Biol.Chem., 269:143-149), 36 nM VIIQ11E33 and PS/PC (25/75, 22 μg/mL).Activity of VIIaQ11E33 was estimated at various time intervals byaddition of 0.15 mm substrate S-2288 (Kabi) and assessing the rate ofp-nitrophenylphosphate product release by absorbance change at 405 nm.Initial activity of the VIIQ11E33 preparation was less than 4% that offully active VIIaQ11E33.

VIIQ11E33 was found to be a much better substrate for activation thanwild-type factor VII. FIG. 2 shows autoactivation of factor VIIQ11E33.The data were analyzed by the relationship in equation 5 (equation 7 ofFiore et al., 1994, supra).ln[VIIa] _(t)=ln[VIIa] ₀ +kcat*y*t  (eq. 5)ln[VIIa]_(t) is the factor VIIa concentration at time t, kcat is thecatalytic rate constant for factor VIIa acting on VII and y is thefractional saturation of VIIa sites. For wild-type factor VIIa, thisrelationship and 1 μM sTF gave a kcat of 0.0045/s and a k_(cat)/K_(m)ratio of 7*10³ M⁻¹s⁻¹. See, Fiore et al., 1994, supra. For the VIIQ11E33enzyme, autoactivation was rapid (FIG. 2) and it was only possible toestimate a lower limit for kcat. This was obtained from the VIIadoubling time of about 25 seconds (kcat=(ln2)/t_(1/2)). The resultingvalue (kcat_(min)=0.03/s), along with the substrate concentration ofthis reaction (3.6*10⁻⁸ M) and the assumption that y=1.0, gave a valuefor kcat/[S]=8*10⁵ M⁻¹s⁻¹. This should be far below the true kcat/Km forVIIaQ11E33, but was about 100-times greater than the value of kcat/Kmfor wild type factor VIIa/sTF estimated by Fiore et al., 1994, supra.Thus, the combination of VIIaQ11E33 enzyme and Factor VIIQ11E33substrate was superior to wild type proteins in the activation step ofcoagulation. This suggested that VIIQ11E33 was superior to wild typeenzyme when coagulation conditions were minimal.

Enhanced activity of VIIaQ11E33: Once generated, factor VIIa activateseither factor X or factor IX. Activation of bovine factor X (0.1 μM) byfactor VIIa was carried out in 50 mM Tris HCl buffer, pH7.5 containing100 mM NaCl, 5 mM calcium, various amounts of phospholipid (PS/PC,25/75) and 1 mg/mL bovine serum albumin at 22.5° C. Factor VIIa (0.06 mMof VIIaQ11E33 or 0.6 nM wild type VIIa) was added at zero time and Xaactivity at 1, 3 and 5 minute time points was determined. Aliquots ofthe reaction mixture (0.2 mL) were mixed with buffer (0.2 mL) containing10 mM EDTA and 0.4 mM S-2222 (Kabi), a chromogenic substrate for factorXa. Absorbance change at 405 nm was determined in a Beckman DU8spectrophotometer. The amount of factor Xa generated was calculated fromthe extinction coefficient (1*10⁴M⁻¹ cm⁻¹) of the p-nitrophenylphosphatereaction product and a velocity of 33/sec for substrate hydrolysis bypurified bovine Xa under the conditions of this assay.

FIG. 3 compares the ability of wild type factor VIIa (open circles) andVIIaQ11E33 (closed circles) to activate factor X in a purified system.Again, VIIaQ11E33 was far superior to wild type factor VIIa in thisreaction. The difference was greatest at low phospholipid concentrationsand diminished to 2-fold at 200 μg phospholipid per mL. This wasexpected from the fact that high membrane concentrations cause a greaterportion of wild type VIIa to bind to the membrane. Once again, theincreased function of VIIaQ11E33 was greatest under conditions of lowphospholipid exposure.

Superior coagulation of VIIaQ11E33: Blood clotting assays were conductedat 37° C. using the hand tilt method to detect clot formation. Humanplasma (0.1 mL) was allowed to equilibrate at 37° C. for 1 minute.Various reagents were added in a volume of 0.1 mL of standard buffer.Soluble tissue factor (50 nM) and phospholipid (PS/PC, 10/90, 75 μg/mL)were added to the plasma, along with the factor VIIa concentration shownin FIG. 4 (0.1-32 nM). Finally, 0.1 mL of 25 mM CaCl₂ was added to startthe reaction. Time to form a clot was measured. In most cases, theaverage and standard deviations of replicate samples was reported.

FIG. 4 shows the coagulation times of wild type VIIa versus VIIaQ11E33in normal human plasma. Coagulation was supported by sTF and addedphospholipid vesicles. Endogenous wild type factor VII is approximately10 nM in concentration, and had virtually no impact on coagulationtimes. The background coagulation was 120 seconds, with or without sTF.Factor VIIaQ11E33 showed approximately 8-fold higher activity than thewild type VIIa under these assay conditions. Similar results wereobtained with factor VIII-deficient plasma, suggesting that the majorpathway for blood clotting in this system involved direct activation offactor X by factor VIIa. Overall, factor VIIaQ11E33 was superior to wildtype VIIa in procoagulant activity supported by membrane vesicles andsoluble tissue factor. Wild type zymogen had virtually no activity underthese conditions, as indicated by similar background clotting times of 2minutes, whether or not sTF was added.

Procoagulant activity with normal tissue factor: Coagulation supportedby normal tissue factor was assayed with standard rabbit brainthromboplastin-HS(HS=high sensitivity) containing calcium (SigmaChemical Co.). This mixture contains both phospholipids andmembrane-bound tissue factor. Rabbit brain thromboplastin-HS was diluted1:100 in buffer and used in the assay of VII (added in the form ofnormal human plasma, which contains 10 nM factor VII) and VIIQ11E33(added as the pure protein). The thromboplastin (0.2 mL) was added toplasma (0.11 mL) to start the reaction and the time required to form ablood clot was measured. Assays were also conducted with full strengththromboplastin, as described by the manufacturer.

At optimum levels of human thromboplastin, wild type VII showed a normallevel of activity, about 1500 units per mg. This is approximately25-fold less than the activity of wild type factor VIIa (80,000 unitsper mg). The VIIQ11E33 gave approximately 1500-3000 units per mg underthe standard assay conditions, only 2-fold greater than wild type VII.

The difference between wild type VII and VIIQ11E33 was much greater whenthe coagulation conditions were sub-optimal. FIG. 5 shows the clottingtimes and zymogen concentrations in assays that contained 0.01-times thenormal thromboplastin level. Under these conditions, VIIQ11E33 wasapproximately 20-fold more active than wild type factor VII. Thus,greater efficacy of the VIIQ11E33 mutant was especially evident whencoagulation conditions were limited, which is relevant to manysituations in vivo.

Anticoagulant Activities of DEGR-VIIaQ11E33: Standard coagulation assayswere performed with normal human serum and human thromboplastin that wasdiluted 1:10 with buffer. The active site of factor VIIaQ11E33 wasmodified by DEGR as described by Sorenson, B. B. et al., 1997, supra.FIG. 6 shows the clotting time of DEGR-VIIaQ11E33 (0-4 nM) incubatedwith thromboplastin, in calcium buffer, for 15 seconds before additionof the plasma. The time to form a clot was determined with the hand tiltmethod. Clotting time was approximately 45 seconds with about 1 nM ofDEGR-VIIaQ11E33. Active-site modified Factor VIIaQ11E33 had enhancedanticoagulation activity.

Example 2 Purification of Factor VII

Factor VII (wild-type or mutant) was purified by Concanavalin A (Con A),DEAE, and affinity chromatography. Crude media of transfected 293 cellswere incubated with Con A resin (Pharmacia) for four hours at 4° C. Theresin then was washed with a solution containing 50 mM Tris, pH7.5, 10mM benzamidine, 1 mM CaCl₂, and 1 mM MgCl₂, and factor VII was elutedwith 0.2 M D-methyl mannoside, 0.5 M NaCl in 50 mM Tris buffer, pH7.5.Factor VII was dialyzed against 50 mM Tris, pH8.0, 50 mM NaCl, 10 mMbenzamidine, and 25 mM D-methyl mannoside overnight.

Dialyzed factor VII then was incubated with DEAE resin (Pharmacia) forone hour and the mixture was packed into a column. The DEAE column waswashed with 50 mM Tris, pH8.0, 10 mM Benzamidine, and 50 mM NaCl, andfactor VII was eluted with a gradient from 50 mM to 500 mM NaCl in 50 mMTris buffer, pH8.0 at a flow rate of 2 mL/min. Fractions containingfactor VII activity were pooled and dialyzed against 50 mM Tris, pH8.0,50 mM NaCl, and 5 mM CaCl₂ overnight. The Con A and DEAE partiallypurified factor VII was activated by bovine factor Xa (weight ratio1:10, Enzyme Research Laboratory) at 37EC for one hour.

Activated factor VII was purified further by affinity chromatography. Acalcium-independent monoclonal antibody for factor VII (Sigma) wascoupled to affigel-10 (Bio-Rad Laboratory) as described by Broze et al.,J. Clin. Invest., 1985, 76:937-946, and was incubated with the affinitycolumn overnight at 4EC. The column was washed with 50 mM Tris, pH7.5,0.1 M NaCl, and factor VIIa was eluted with 50 mM Tris, pH7.5, 3 M NaSCNat a flow rate of 0.2 mL/min. The eluted fractions were immediatelydiluted five fold into 50 mM Tris, pH7.5, 0.1 M NaCl. Fractionscontaining factor VIIa activity were pooled, concentrated, and dialyzedagainst 50 mM Tris, pH7.5, 0.1 M NaCl overnight.

The protein concentration of factor VIIa was determined with a Bio-Radprotein assay kit, using BSA as the standard. The purity of factor VIIawas assayed by Coomassie gel and Western Blotting under reduced anddenatured conditions. Proteolytic activity of factor VIIa was measuredusing a synthetic peptide substrate spectrozyme-FVIIa (AmericanDiagnostica) in the presence of thromboplastin (Sigma). Purified factorVIIa was stored in 0.1 mg/ml BSA, 0.1 M NaCl, 50 mM Tris, pH7.5 at−80EC.

Procoagulant effectiveness of factor VIIa mutants was assessed usingstandard in vitro clotting assays (and modifications thereof);specifically, the prothrombin time (PT) assay and the activated partialthromboplastin (aPTT) assay. Various concentrations of Factor VIIamutants were evaluated in pooled normal human donor plasma and incoagulation factor-deficient (Factor VIII, Factor IX, Factor VII) humanplasmas (Sigma). Clotting times were determined at 37EC using both aFibroSystem fibrometer (BBL) with a 0.3 mL probe and a Sysmex CA-6000Automated Coagulation Analyzer (Dade Behring).

Platelet poor plasma (PPP) was prepared from pooled normal human donorblood. Blood (4.5 mLs) was drawn from each healthy donor into citrated(0.5 mLs of 3.2% buffered sodium citrate) Vacutainer tubes. Plasma wasobtained after centrifugation at 2,000 g for ten minutes and was kept onice prior to use. Purchased factor-deficient plasmas were reconstitutedaccording to the manufacturer's instructions. Serial dilutions from eachstock of Factor VIIa mutant were all prepared in plasma. All plasmas(with or without Factor VIIa mutants) were kept on ice prior to use. Inall cases, the time to form a clot was measured. The average andstandard deviation of replicate samples was reported.

The prothrombin time (PT) assay was performed in plasmas (with orwithout serial dilutions of added FVIIa mutants) using either of thefollowing PT reagents: Thromboplastin C-Plus (Dade), Innovin (Dade),Thromboplastin With Calcium (Sigma), or Thromboplastin HS With Calcium(Sigma). Assays were conducted according to manufacturer's instructions.In addition to using PT reagents at the manufactured full-strengthconcentration, the PT assay also was performed using various dilutionsof PT reagent. In all cases, the time to form a clot was measured. Theaverage and standard deviation of replicate samples was reported.

The activated partial thromboplastin (aPTT) assay was performed inplasmas (with or without serial dilutions of added Factor VIIa mutants)using either Actin FS (Dade) or APTT reagent (Sigma). Clotting wasinitiated using 0.025M CaCl₂ (Dade) for Actin FS (Dade) or 0.02M CaCl₂(Sigma) for APTT reagent (Sigma). Assays were conducted according tomanufacturer's instructions. In addition to using aPTT reagents at themanufactured full-strength concentration, the aPTT assay also wasperformed using various dilutions of aPTT reagent. In all cases, thetime to form a clot was measured. The average and standard deviation ofreplicate samples was reported.

Clotting assays also were conducted at 37° C. in which differentconcentrations of phospholipid vesicles [at varying ratios of PS/PC orPS/PC/PE (PE=phosphatidylethanolamine)] were added to plasmas (with orwithout serial dilutions of added FVIIa mutants). Clotting was initiatedby the addition of 20 mM CaCl₂. Various reagents were added in standardbuffer. In all cases, the time to form a clot was measured. The averageand standard deviation of replicate samples was reported.

Specific Factor VIIa clotting activity was assessed in plasmas (with orwithout added Factor VIIa mutants) using the STACLOT VIIa-rTF kit(Diagnostica Stago), as per the manufacturer's instructions. This kit isbased on the quantitative clotting assay for activated FVII (Morrisseyet al., 1993, Blood, 81(3):734-744). Clotting times were determinedusing both a FibroSystem fibrometer (BBL) with a 0.3 mL probe and aSysmex CA-6000 Automated Coagulation Analyzer (Dade Behring).

Example 3 Circulatory Time of Factor VIIQ11E33 in the Rat

Two anesthetized (sodium nembutol) Sprague Dawley rats (325-350 g) wereinjected with 36 μg of factor VIIQ11E33 at time zero. Injection wasthrough the juggler vein, into which a cannula had been placed. At thetimes shown in FIG. 7, blood was withdrawn from the carotid artery, intowhich a cannula had been inserted by surgery. The amount of factorVIIQ11E33 in the circulation was estimated from the clotting time ofhuman factor VII-deficient plasma, to which 1 μL of a 1:10 dilution ofthe rat plasma was added. A 1:100 dilution of rabbit brainthromboplastin-HS (Sigma Chemical Co.) was used. Coagulation wasassessed by the manual tube tilt method as described in Example 1. Theamount of factor VII activity in the plasma before injection ofVIIQ11E33 was determined and was subtracted as a blank. Theconcentration of factor VIIQ11E33 in the circulation is given as log nM.A sham experiment in which a third animal received the operation andcannulation but no factor VIIQ11E33 was conducted. The amount of factorVII activity in that animal did not change over the time of theexperiment (100 minutes). At the end of the experiment, the animals wereeuthanized by excess sodium nembutol.

The rats appeared normal throughout the experiment with no evidence ofcoagulation. Therefore, the factor VIIQ11E33 did not causeindiscriminate coagulation, even in the post-operative rat. Thecirculation life-time of the VIIQ11E33 was normal (FIG. 7), withapproximately 40% of the protein being cleared in about 60 minutes andan even slower disappearance of the remaining protein. This was similarto the rate of clearance of bovine prothrombin from the rat. Nelsestuenand Suttie, 1971, Biochem. Biophys. Res. Commun., 45:198-203. This issuperior to wild-type recombinant factor VIIa that gave a circulationhalf-time for functional assays of 20-45 minutes. Thomsen, M. K., etal., 1993, Thromb. Haemost., 70:458-464. This indicated that factorVIIQ11E33 was not recognized as an abnormal protein and that it was notrapidly destroyed by coagulation activity. It appeared as a normalprotein and should have a standard circulation lifetime in the animal.

Example 4 Enhancement of the membrane site and activity of protein C

Bovine and human protein C show a high degree of homology in the aminoacids of their GLA domains (amino terminal 44 residues), despite about10-fold higher membrane affinity of the human protein. Bovine protein Ccontains a proline at position 11 versus a histidine at position 11 ofhuman protein C. The impact of replacing proline-11 in bovine protein Cwith histidine, and the reverse change in human protein C, was examined.In both cases, the protein containing proline-11 showed lower membraneaffinity, about 10-fold for bovine protein C and 5-fold for humanprotein C. Activated human protein C (hAPC) containing proline atposition 11 showed 2.4 to 3.5-fold lower activity than wild type hAPC,depending on the assay used. Bovine APC containing histidine-11displayed up to 15-fold higher activity than wild type bAPC. Thisdemonstrated the ability to improve both membrane contact and activityby mutation.

Mutagenesis of Protein C: A full-length human protein C cDNA clone wasprovided by Dr. Johan Stenflo (Dept. of Clinical Chemistry, UniversityHospital, Malmö, Sweden). The bovine protein C cDNA clone was providedby Dr. Donald Foster (ZymoGenetics, Inc., USA). The GenBank accessionnumber for the nucleotide sequence of bovine protein C is KO2435, NIDg163486 and is KO2059, NID A190322 for the nucleotide sequence of humanprotein C.

Site-directed mutagenesis was performed by a PCR method. For humanprotein C mutagenesis of histidine-11 to proline, the followingoligonucleotides were synthesized: A, 5′-AAA TTA ATA CGA CTC ACT ATA GGGAGA CCC AAG CTT-3′ (corresponding to nucleotides 860-895 in the vectorpRc/CMV, SEQ ID NO:7) to create a Hind III site between pRc/CMV andprotein C. B, 5′-GCA CTC CCG CTC CAG GCT GCT GGG ACG GAG CTC CTC CAGGAA-3′(corresponding to the amino acid residues 4-17 in human protein C,the 8th residue in this sequence was mutated from that for human proteinC to that of bovine protein C, as indicated by the underline, SEQ IDNO:8).

For bovine protein C mutagenesis of proline-11 to histidine, thefollowing oligonucleotides were synthesized: A, (as described above); C,5′-ACG CTC CAC GTT GCC GTG CCG CAG CTC CTC TAG GAA-3′ (corresponding toamino acid residues 4-15 in bovine protein C, the 6th amino acid wasmutated from that for bovine protein C to that of human protein C asmarked with underline SEQ ID NO:9); D, 5′-TTC CTA GAG GAG CTG CGG CACGGC AAC GTG GAG CGT-3′ (corresponding to amino acid residues 4-15 inbovine protein C, the 7th amino acid was mutated from that for bovineprotein C to that of human protein C; mutated nucleotides areunderlined, SEQ ID NO:10); E, 5′-GCA TTT AGG TGA CAC TAT AGA ATA GGG CCCTCT AGA-3′ (corresponding to nucleotides 984-1019 in the vectorpRc/CMV), creating a Xba I site between pRc/CMV and protein C, SEQ IDNO:11).

Both human and bovine protein C cDNAs were cloned into the Hind III andXba I sites of the expression vector pRc/CMV. Human protein C cDNAcontaining the 5′ terminus to amino acid-17 was PCR amplified withintact human protein C cDNA and primers A and B. The volume for the PCRreaction was 100 μl and contained 0.25 μg of template DNA, 200 μM eachof the four deoxyribonucleoside triphosphates, 0.5 mM of each primer and2.5 U of Pwo-DNA polymerase (Boehringer Mannheim) in Tris-HCl buffer (10mM Tris, 25 mM KCl, 5 mM (NH₄)₂SO₄, and 2 mM MgSO₄, pH8.85). Sampleswere subjected to 30 cycles of PCR consisting of a 2 minute, 94° C.denaturation period, a 2 minute, 55° C. annealing period, and a 2minute, 72° C. elongation period. After amplification, the DNA waselectrophoresed through an 0.8% agarose gel in 40 mM Tris-acetate buffercontaining 1 mM EDTA. PCR products were purified with JET PlasmidMiniprep-Kit (Saveen Biotech AB, Sweden). Human protein C cDNAcontaining respective mutations was cleaved by Hind III and Bsr BI, andthen cloned into pRc/CMV vector that was cleaved by Hind III/Xba I andthat contained human protein C fragment from Bsr BI to the 3′ terminusto produce a human protein C full length cDNA with the mutation.

Bovine protein C cDNA, containing the 5′ terminus through amino acid-11,was PCR amplified with intact human protein C cDNA and primers A and C.Bovine protein C cDNA from amino acid 11 to the 3′ terminus wasamplified with intact human protein C cDNA and primers D and E. Thesetwo cDNA fragments were used as templates to amplify full length bovineprotein C cDNA containing mutated amino acids with primers A and E. PCRreaction conditions were identical to those used for HAPC. The bovineprotein C cDNA containing the respective mutations was cleaved by HindIII and Bsu 36I, and the Hind III/Bsu36I fragment was cloned intopRc/CMV vector containing intact bovine protein C fragments from the Bsu36I to the 3′ terminus to produce full-length bovine protein C cDNAcontaining the mutation. All mutations were confirmed by DNA sequencingprior to transfection.

Cell Culture and Expression: The adenovirus-transfected human kidneycell line 293 was grown in DMEM medium supplemented with 10% fetal calfserum, 2 mM L-glutamine, 100 U/ml of penicillin, 100 U/ml streptomycinand 10 μg/ml vitamin K₁. Transfection was performed using the lipofectinmethod. Felgner, P. L. et al., 1987, Proc. Natl. Acad. Sci. USA,84:7413-7417. Two μg of DNA was diluted to 0.1 ML with DMEM containing 2mM of L-glutamine medium. Ten μL of Lipofectin (1 mg/ml) was added to100 μL of DMEM containing 2 mM L-glutamine medium. DNA and lipofectinwere mixed and left at room temperature for 10-15 min. Cell monolayers(25-50% confluence in 5-cm petri-dishes) were washed twice in DMEM with2 mM L-glutamine medium. The DNA/lipid mixture was diluted to 1.8 mL inDMEM containing 2 mM L-glutamine medium, added to the cells andincubated for 16 hours. The cells were fed with 2 mL of complete mediumcontaining 10% calf serum, left to recover for another 48-72 hours andthen trypsinized and seeded into 10-cm dishes with selection medium(DMEM containing 10% serum and 400 μg/mL of Geneticin) at 1:5. Yan, S.C. B. et al., 1990, Bio/Technology 655-661. Geneticin-resistant colonieswere obtained after 3-5 weeks of selection. Twenty four colonies fromeach DNA transfection were picked, grown to confluence and the mediascreened for protein C expression with a dot-blot assay using monoclonalantibody HPC4 (for human protein C) and monoclonal antibody BPC5 (forbovine protein C). Clones producing high amounts of protein wereisolated and grown until confluence in the presence of 10 μg/mL ofvitamin K₁.

The purification of bovine recombinant protein C and its mutant werebased on the method described previously with some modifications.Rezair, A. R., and Esmon, C. T., 1992, J. Biol. Chem., 267:26104-26109.Conditioned serum-free medium from stably transfected cells wascentrifuged at 5000 rpm at 4° C. for 10 minutes. The supernatant wasfiltered through 0.45 μm of cellulose nitrate membranes (MicroFiltration Systems, Japan). EDTA (5 mM, final concentration) and PPACK(0.2 μM, final concentration) were added to the conditioned medium from293 cells, then passed through a Pharmacia FFQ anion-exchange column atroom temperature using Millipore Con Sep LC100 (Millipore, USA). Theprotein was eluted with a CaCl₂ gradient (starting solution, 20 mMTris-HCl/150 mM NaCl, pH7.4; limiting solution, 20 mM Tris-HC/150 mMNaCl/30 mM CaCl₂, pH7.4). After removal of the CaCl₂ by dialysis andChelex 100 treatment, the protein was reabsorbed to a second FFQ column,then eluted with an NaCl gradient (starting solution 20 mM Tris-HCl/150mM NaCl, pH7.4; limiting solution, 20 mM Tris-HCl/500 mM NaCl, pH7.4).At this point in the purification, wild-type and the mutant recombinantbovine protein C were homogeneous as determined by SDS-PAGE.

The first column used for purification of wild-type and mutantrecombinant human protein C was the same as that described for bovineprotein C. The chromatographic method described by Rezair and Esmon wasemployed with some modifications described for the method of protein Spurification. Rezair, A. R., and Esmon, C. T., 1992, supra; He, Z. etal., 1995, Eur. J. Biochem., 227:433-440. Fractions containing protein Cfrom anion-exchange chromatography were identified by dot-blot. Positivefractions were pooled and applied to an affinity column containing theCa²⁺-dependent antibody HPC-4. The column was equilibrated with 20 mMTris-HCl, 150 mM NaCl, pH7.4, containing 5 mM Benzamidine-HCl and 2 mMCaCl₂. After application, the column was washed with the same buffercontaining 1 M NaCl. Protein C was then eluted with 20 mM Tris-HCl, 150mM NaCl and 5 mM EDTA, pH7.4, containing 5 mM Benzamidine-HCl. Afterpurification, the purity of all human and bovine recombinant protein Cpreparations was estimated by SDS-PAGE followed by silver staining.Proteins were concentrated using YM 10 filters (Amicon), then dialyzedagainst buffer (50 mM Tris-HCl and 150 mM NaCl, pH7.4) for 12 hours andstored at −70° C. The concentrations of proteins were measured byabsorbance at 280 nm.

Association of normal and mutant protein C molecules with membranes:LUVs and SUVs were prepared by methods described in Example 1. Lightscattering at 90° to the incident light was used to quantitateprotein-membrane binding as described above for Factor VII (25 μg/mL ofPS/PC, (25/75) at 5 mM calcium (0.05 M Tris buffer-0.1 M NaCl, pH7.5).

Bovine protein C containing histidine at position 11 interacted withmembranes with about 10-fold higher affinity than wild type protein.When fit to equation 2, the data gave K_(D) values of 930±80 nM forprotein C-H11 and 9200±950 nM for wild type protein C (FIG. 8A). Thedifference in affinity corresponded to about 1.4 kcal/mol at 25° C. Infact, membrane affinity of bovine protein C-H11 was almost identical tothat of native human protein C (660 nM, FIG. 8B). This suggested thatproline 11 formed a major basis for differences between the membranebinding site of human and bovine proteins.

The reverse substitution, replacement of His-11 of human protein C byproline, decreased membrane affinity (FIG. 8B). When fit to equation 2,these data gave K_(D) values of 660±90 nM for wild type human protein Cand 3350±110 nM for human protein C-P11. The impact of prolineintroduction was only slightly less than that of proline in the bovineproteins.

Impact of proline-11 on activity of activated protein C: Activatedprotein C was generated by thrombin cleavage, using identical conditionsfor both the wild type and mutant proteins. Approximately 150 μg of thevarious protein C preparations (1 mg/mL) were mixed with bovine thrombin(3 μg) and incubated at 37° C. for 5 hours. The reaction product wasdiluted to 0.025 M Tris buffer-0.05 M NaCl and applied to a one mLcolumn of SP-Sephadex C-50. The column was washed with one mL of thesame buffer and the flow-through was pooled as activated protein C.Approximately 65-80% of the protein applied to the column was recovered.APC activity was determined by proteolysis of S2366 (0.1 mM) at 25° C.The preparations were compared to standard preparations obtained onlarger scale. Standard human APC was provided by Dr. Walter Kisiel. Forbovine proteins, the standard was a large-scale preparation ofthrombin-activated APC. The activity of bovine APC was consistent forall preparations of normal and mutant proteins (±5%). Two preparationsof bovine APC were used for comparisons. Human APC generated fromthrombin was 55 to 60% as active as the standard. The concentrationsreported in this study were based on activity toward S2366, relative tothat of the standard.

Standard APTT test used bovine or human plasma and standard APTT reagent(Sigma Chemical Co.) according to manufacturers instructions.Alternatively, phospholipid was provided in the form of vesicles formedfrom highly purified phospholipids. In this assay, bovine plasma (0.1mL) was incubated with either kaolin (0.11 mL of 5 mg/mL in 0.05 M Trisbuffer, 0.1 M NaCl, pH7.5) or ellagic acid (0.1 mM in buffer) for 5minutes at 35° C. Coagulation was started by adding 0.1 mL of buffercontaining phospholipid and the amounts of APC shown, followed by 0.1 mLof 25 mM calcium chloride. All reagents were in standard buffercontaining 0.05 M Tris buffer, 0.1 M NaCl, pH7.5. An average of a14-fold higher concentration of wild type bAPC was needed to duplicatethe impact of the H11 mutant. Coagulation time at 10 nM bAPC-H11 wasgreater than 120 minutes. Standard APTT reagent (Sigma Chemical Co.)gave a clotting time of about 61 seconds at 35° C. with this plasma.Time required to form a clot was recorded by manual technique. Theamount of phospholipid was designed to be the limiting component in theassay and to give the clotting times shown. The phospholipids used wereSUVs (45 μg/0.4 mL in the final assay, PS/PC, 10/90) or LUVs (120 μg/0.4mL in the final assay, PS/PC, 25/75).

The anticoagulant activity of activated protein C was tested in severalassays. FIG. 9 shows the impact on the APTT assay, conducted withlimiting phospholipid. Under the conditions of this assay, coagulationtimes decreased in a nearly linear, inverse relationship withphospholipid concentration. Approximately 14-times as much wild typebovine APC was needed to equal the effect of bovine APC-H11.

Parts of the study in FIG. 9 were repeated for membranes of PS/PC(25/75, LUV). Again, activity was limited by phospholipid, and itsconcentration was adjusted to give a control clotting time of 360seconds (120 μg of 25% PS in the 0.4 mL assay). Approximately 15-foldmore wild type enzyme was needed to equal the impact of the H11 mutant.Finally, standard APTT reagent (Sigma Chemical Co., standard clottingtime 50±2 seconds) was used. Approximately 10.0±0.7 nM of wild typeenzyme was needed to double the coagulation time to 102±5 seconds. Thesame impact was produced by 2.2±0.1 nM bovine APC-H11. Phospholipid wasnot rate limiting in the standard assay so a smaller impact on membraneaffinity may be expected.

Results for human proteins are shown in FIG. 8B. About 2.5 times as muchhuman APC containing proline-11 was required to prolong coagulation tothe extent of wild type APC. A lower impact of proline-11 introductionmay reflect the smaller differences in membrane affinity of the humanproteins (FIG. 9B).

Inactivation of factor Va: Factor Va inactivation was assayed by themethod of Nicolaes et al., 1996, Thrombosis and Haemostasis, 76:404-410.Briefly, for bovine proteins, bovine plasma was diluted 1000-fold by0.05 M Tris, 0.1 M NaCl, 1 mg/mL bovine serum albumin and 5 mM calciumat pH7.5. Phospholipid vesicles (5 μg/0.24 mL assay) and 5 μL of 190 nMthrombin were added to activate factor V. After a 10-minute incubationat 37° C., APC was added and the incubation was continued for 6 minutes.Bovine prothrombin (to 10 μM final concentration) and factor Xa (0.3 nMfinal concentration) were added and the reaction was incubated for oneminute at 37° C. A 20 μL sample of this activation reaction was added to0.38 mL of buffer (0.05 M Tris, 0.1 M NaCl, 5 mM EDTA, pH7.5) containingS2288 substrate (60 μM). The amount of thrombin was determined by thechange in absorbance at 405 nM (ε=1.0*10⁴ M⁻¹s⁻¹, k_(cat) forthrombin=100/s). For human proteins, human protein S-deficient plasma(Biopool Canada, Inc.) was diluted 100-fold, factor Va was activated byhuman thrombin and the factor Va produced was assayed with the reagentsused for the bovine proteins.

Bovine APC-H11 was 9.2-fold more active than wild type (FIG. 10A) ininactivating factor Va. As for membrane binding (above), the impact ofproline-11 was less with the human proteins, with an average of 2.4-folddifference between the curves drawn for wild type and P-11 mutant (FIG.10B). Similar results were obtained with normal human plasma.

Example 5 Identification of an Archetype Membrane Affinity for theMembrane Contact Site of Vitamin K-Dependent Proteins

Comparison of various human and bovine protein C mutants and othervitamin K-dependent polypeptides led to a proposed membrane contact sitearchetype. The electrostatic archetype consists of an electropositivecore on one surface of the protein, created by bound calcium ions,surrounded by a halo of electronegative charge from amino acids of theprotein. The closer a member of this protein family approaches thiselectrostatic pattern, the higher its affinity for membranes.

Phospholipid vesicles, wild type bovine protein C, protein-membraneinteraction studies, activation and quantitation of protein C, andactivity analysis were as described in Example 4.

Recombinant, mutant protein C was generated by the following procedures.Site-directed mutagenesis was performed by a PCR method. The followingoligonucleotides were synthesized: A, as described in Example 4; F,5′-GCA TTT AGG TGA CAC TAT AGA ATA GGG CCC TCT AGA-3′ (corresponding tonucleotides 984-1019 in the vector pRc/CMV, SEQ ID NO:11), creating aXba I site between pRc/CMV and protein C; G, 5′-GAA GGC CAT TGT GTC TTCCGT GTC TTC GAA AAT CTC CCG AGC-3′ (corresponding to amino acid residues40-27 in bovine protein C, the 8th and 9th amino acids were mutated fromQN to ED as marked with underline, SEQ ID NO:12); H, 5′-CAG TGT GTC ATCCAC ATC TTC GAA AAT TTC CTT GGC-3′ (corresponding to amino acid residues38-27 in human protein C, the 6th and 7th amino acids in this sequencewere mutated from QN to ED as indicated with the underline, SEQ IDNO:13); I, 5′-GCC AAG GAA ATT TTC GAA GAT GTG GAT GAC ACA CTG-3′(corresponding to amino acid residues 27-38 in human protein C, the 6thand 7th amino acids in this sequence were mutated from QN to ED asindicated with underline, SEQ ID NO:14); J, 5′-CAG TGT GTC ATC CAC ATTTTC GAA AAT TTC CTT GGC-3 (corresponding to amino acid residues 38-27 inhuman protein C, the 7th amino acids in this sequence were mutated fromQ to E as indicated with underline, SEQ ID NO:15); K, 5′-GCC AAG GAA ATTTTC GAA AAT GTG GAT GAC ACA CTG-3′ (corresponding to amino acid residues27-38 in human protein C, the 6th amino acid in this sequence wasmutated from Q to E as indicated with underline, SEQ ID NO:16);

-   -   Both bovine and human protein C full length cDNAs were cloned        into the Hind III and Xba I site of the vector pRc/CMV. To        obtain bovine protein C mutant E33D34, PCR amplification of the        target DNA was performed as follows. Bovine protein C cDNA        containing the 5′ terminus to the amino acid at position 40, was        amplified with intact bovine protein C cDNA and primers A and C.        The PCR reaction conditions were as described in Example 3. The        sample was subjected to 30 cycles of PCR consisting of a 2 min        denaturation period at 94° C., a 2 min annealing period at        55° C. and a 2 min elongation period at 72° C. After        amplification, the DNA was electrophoresed through an 0.8%        agarose gel in 40 mM Tris-acetate buffer containing 1 mM EDTA.        The PCR products were purified with The Geneclean III kit (BIO        101, Inc. USA), and the PCR fragment of bovine protein C cDNA        containing the respective mutations was cleaved by Hind III and        Bbs I. The Hind III/Bbs I fragment and the human protein C        fragment (Bbs 1-3′ terminus) were cloned into the Hind III and        Xba I sites of pRc/CMV vector to produce a full-length bovine        protein C cDNA with the mutations. Bovine protein C mutant H11        E33 D34 was created in the same way, but used bovine protein C        mutant H11 as a template in the PCR reaction.

Human protein C cDNA containing the 5′ terminus to amino acid-38 was PCRamplified with intact human protein C cDNA and primers A and D. Humanprotein C cDNA from amino acid 27 to the 3′ terminus was amplified withintact human protein C cDNA and primers B and E. These two cDNAfragments were used as templates to amplify full length bovine protein CcDNA containing mutated amino acids (E33 D34) with primers A and B.Human protein C mutant E33 was obtained by the following steps: humanprotein C cDNA containing the 5′ terminus to amino acid 38 was amplifiedwith intact human protein C cDNA and primers A and F. Human protein CcDNA from amino acid 27 to the 3′ terminus was amplified with intacthuman protein C cDNA and primers B and G. These two cDNA fragments wereused as templates to amplify full length bovine protein C cDNAcontaining mutated amino acids (E33) with primers A and B. The PCRmixture and program were described above. The human protein C PCRproducts containing respective mutations were cleaved by Hind III andSal I, and then the fragment (Hind III-Sal I) together with intact humanprotein C fragment (Sal I-3′ terminus) were cloned into the Hind III andXba I sites of pRc/CMV vector to produce the full length human protein CcDNA with the respective mutations. Human protein C containing a glycineresidue at position 12, in conjunction with the E33D34 mutations alsowas made. All mutations were confirmed by DNA sequencing prior totransfection.

The adenovirus-transfected human kidney cell line 293 was cultured andtransfected as described in Example 4. Bovine and human recombinantprotein C and mutants were purified as described in Example 4.

The vitamin K-dependent proteins were classified into four groups on thebasis of their affinities for a standard membrane (Table 5). Sequencesof the amino terminal residues of some relevant proteins including humanprotein C (hC, SEQ ID NO:1), bovine protein C (bC, SEQ ID NO:2), bovineprothrombin (bPT, SEQ ID NO:17), bovine factor X (bX, SEQ ID NO:18),human factor VII (hVII, SEQ ID NO:3), human protein Z (hZ, SEQ IDNO:20), and bovine protein Z (bZ, SEQ ID NO:21) are given for reference,where X is Gla (γ-carboxyglutamic acid) or Glu. TABLE 5 bPT:ANKGFLXXVRK₁₁GNLXRXCLXX₂₁PCSRXXAFXA₃₁LXSLSATDAF₄₁ WAKY bX:ANS-FLXXVKQ₁₁GNLXRXCLXX₂₁ACSLXXARXV₃₁FXDAXQTDXF₄₁ WSKY hC:ANS-FLXXLRH₁₁SSLXRXCIXX₂₁ICDFXXAKXI₃₁FQNVDDTLAF₄₁ WSKH bC:ANS-FLXXLRP₁₁GNVXRXCSXX₂₁VCXFXXARXI₃₁FQNTXDTMAF₄₁ WSFY hVII:ANA-FLXXLRP₁₁GSLXRXCKXX₂₁QCSFXXARXI₃₁FKDAXRTKLF₄₁ WISY hZ:AGSYLLXXLFX₁₁GNLXKXCYXX₂₁ICVYXXARXV₃₁FXNXVVTDXF₄₁ WRRY bZ:AGSYLLXXLFX₁₁GHLXKKCWXX₂₁ICVYXXARXV₃₁FXDDXTTDXF₄₁ WRTY Charges andAffinity Residue 11 + 29 + 33 + 34 = Sum Total K_(D) (nM) Class I bZ−2 + −2 −1 −4 −6 0.2^(a) − 32  hZ −2 + −2 −3 −5 2.0^(a) − 170 Class IIbPT-TNBS −2 −2 −1 <10 hVII-Q11E33 + −2 −1 −2 −2 10 hS + −2 −1 −2 −2 40bX + −2 −1 −2 −3 40 bC-E33D34 P + −2 −1 −2 −4 125 hX + + −2 −1 −1 −2 160bPT + −2 −1 0 100 hPT + −2 −1 −1 — bS + + −2 0 0 120 Class III bIX + −2−1 −1 1000 hIX + −2 −1 −1 1000 hC + +1 −2 660 bC-H11 + +1 −1 930Class IV hC P + +1 −2 3300 hVII P + + −1 +1 +1 4000 bC P + +1 −1 9200bvII P + + +1 0 15000^(a)Higher affinity value equals k_(dissociation)/1*10⁷M⁻¹s⁻¹; thedenominator is a typical k_(association) for other proteins.

In Table 5, vitamin K-dependent polypeptide mutants are in bold. Thetotal charge (residues 1-34) includes 7 calcium ions (+14) and the aminoterminus (+1).

Protein Z was assigned to class I on the basis of its dissociation rateconstant, which was 100 to 1000 times slower than that of otherproteins. If protein Z displayed a normal association rate constant(about 10⁷ M⁻¹ s⁻¹) the K_(D) would be about 10⁻¹⁰ M. Wei, G. J. et al.,1982, Biochemistry, 21:1949-1959. The latter affinity may be the maximumpossible for the vitamin K proteins. Protein Z is a candidate foranticoagulation therapy as a cofactor for inhibition of factor Xa by theprotein Z-dependent protease inhibitor (ZPI). Incubation of protein Zand ZPI with factor Xa reduces the procoagulant activity of factor Xa.See, for example, Han et al., Proc. Natl. Acad. Sci. USA, 1998,95:9250-9255. Enhancement of association kinetics would speed inhibitionrates and increase binding affinity at equilibrium. Protein Z containsgly-2, which is thought to abolish interaction of asn-2 with boundcalcium in other proteins. The presence of a glycine residue at position2 may destabilize protein folding and lower association kinetics. Inaddition, the lower affinity of protein Z relative to that of bovineprotein Z may arise from fewer anionic charges in positions 34-36 (Table5).

Class IV proteins differed from class III in the presence of proline-11,which may alter affinity by non-electrostatic means.

While a relatively weak correlation existed between membrane affinityand net negative charge on residues 1-34, an excellent correlation wasfound when only residues 5, 11, 29, 33 and 34 were considered (Table 5).The latter amino acids are located on the surface of the protein. Anumber of proteins were modeled by amino acid substitution into theprothrombin structure and their electrostatic potentials were estimatedby the program DelPhi. A sketch patterned after the electrostaticpotential of bovine protein Z is shown in FIG. 11. Electronegative sitesat 7, 8, 26, 30, 33, 34 and 11 produce a halo of electronegative chargesurrounding a cationic core produced by the calcium-lined pore (FIG.11). The closer a protein structure approaches this structure, thehigher its affinity for the membrane. The highest affinity proteins showan electronegative charge extending to amino acids 35 and 36. Thiscorrelation is apparent from the wild-type proteins, the mutants andchemically modified proteins.

The pattern for other structures can be extrapolated from examination ofthe charge groups that are absent in other proteins. For example, Lys-11and Arg-10 of bovine prothrombin generate high electropositive regionsin their vicinity; the lack of Gla-33 in protein C and Factor VII createless electronegativity in those protein regions. In all cases, highestaffinity corresponded to a structure with an electropositive core thatwas completely surrounded by electronegative protein surface, as shownfor protein Z. The exceptions to this pattern are the proteins withPro-11, which may lower affinity by a structural impact and ser-12(human protein C), which is a unique uncharged residue.

To further test the hypothesis of an archetype for electrostaticdistribution, site-directed mutagenesis was used to replace Gln33Asn34of bovine and human protein C with Glu33Asp34. Glu33 should be furthermodified to Gla during protein processing. These changes altered theelectrostatic potential of bovine protein C to that of bovine factor X.The membrane affinity of the mutant protein was expected to be lowerthan that of factor X due to the presence of proline-11. Indeed, thebovine protein C mutant gave a membrane affinity similar to that ofbovine prothrombin (FIG. 12A), and slightly less than that of bovinefactor X (Table 5).

More interesting was that clot inhibition by APC was greater for themutant than for the wild type enzyme (FIG. 12B, C). Inclusion of resultsfor the P 11 H mutant of bovine protein C from Example 3 showed that afamily of proteins could be produced, each with different membraneaffinity and activity, by varying the amino acid substitutions atpositions 11, 33 and 34.

Human protein C mutants containing E33 and E33D34 resulted in a smallincrease in membrane binding affinity (FIG. 13 a). Activity of thesemutants was slightly less than the wild-type enzyme (FIG. 13 b). Resultswith mutants of bovine protein C suggest that failure of the E33D34mutation in the human protein may arise from H11 and/or other uniqueamino acids in the protein. FIG. 14A shows that the H11 mutant of bovineprotein C bound to the membrane with about 10-fold higher affinity thanwild type protein, the E33D34 mutant bound with about 70-times theaffinity, but that the triple mutant, H11E33D34, was only slightlybetter than the H11 mutant. This relationship was mirrored in theactivity of APC formed from these mutants (FIG. 14B). This resultsuggested that the presence of H11 reduced the impact of E33D34 onmembrane binding affinity.

These results indicated that introduction of E33D34 alone may not beoptimal for all proteins. Consequently, other mutations may be desirableto create human protein C that will use E33D34 and have maximumincreased membrane affinity. The result with the bovine proteinsuggested that histidine 11 may be the primary cause of this phenomenon.Consequently, H11 may be altered to glutamine or to another amino acidin human protein C, along with the E33D34 mutation. Another amino acidthat may impact the affinity is the serine at position 12, an amino acidthat is entirely unique to human protein C. These additional changesshould produce proteins with enhanced membrane affinity. Substitution ofa glycine residue for serine at position 12 of human activated proteinC, in conjunction with E33D34 resulted in 9-fold higher activity thanwild-type activated human protein C. Activity of the G12E33D34 mutantwas assessed with a dilute thromboplastin assay using a control clottingtime of 30 seconds and normal human plasma. The electrostatic archetypealso was tested by comparison of human and bovine factor X. The presenceof lysine-11 in human factor X suggests that it should have loweraffinity than bovine factor X. This prediction was borne out, by theresult shown in FIG. 15.

Earlier studies had shown that trinitrobenzenesulfonic acid (TNBS)modification of bovine and human prothrombin fragment 1 had relativelylittle impact (0 to 5-fold) on membrane affinity. Weber, D. J. et al.,1992, J. Biol. Chem., 267:4564-4569; Welsch, D. J. et al., 1988,Biochemistry, 27:4933-4938. Conditions used for the reaction resulted inderivatization of the amino terminus, a change that is linked to loweredmembrane affinity. Welsch, D. J. and Nelsestuen, G. L., 1988,Biochemistry, 27:4939-4945. Protein modification in the presence ofcalcium, which protects the amino terminus, resulted in TNBS-modifiedprotein with much higher affinity for the membrane than native fragment1.

The suggestion that protein Z constitutes the archetype was based on itsdissociation rate constant and that a normal association rate wouldgenerate a K_(D)=10⁻¹⁰ M. Whether this value can be reached isuncertain. It is possible that the slow association rate of protein Z iscaused by improper protein folding, resulting in a low concentration ofthe membrane-binding conformation. If conditions can be altered toimprove protein folding, association rates of protein Z should improve.Indeed, the association rate constant for protein Z was improved byalteration of pH. The basis for this observation may be related to anunusual feature of the prothrombin structure, which is the closeplacement of the amino terminus (+1 at pH7.5) to calcium ions 2 and 3.The +1 charge on the amino terminus is responsible for the slightelectropositive region just above Ca-1 in FIG. 11. Charge repulsionbetween Ca and the amino terminal may destabilize protein folding andcould be a serious problem for a protein that had low folding stability.

Table 6 provides additional support for the archetype model. It showsthe relationship between distance of ionic groups from strontium ions 1and 8 (corresponding to calcium 1 and an extra divalent metal ion foundin the Sr x-ray crystal structure of prothrombin). The pattern suggeststhat the closer an ionic group is to these metal ions, the higher itsimpact on membrane affinity. The exception is Arg-16, which contributesto the charge of the electropositive core. Higher affinity is correlatedwith electronegative charge at all other sites. This correlation alsoapplies to the GLA residues. TABLE 6 Distance to Sr-1, 8 and IonImportance. Distance (Å) to: Impact/ion Position Atom^(a) Sr-1 Sr-8 onK_(D)(K_(M)) (A.A-Protein)  3(K-PT) ε-N 22.1 21.7 Low or Unknown 5(K-IX) para-C(F) 20.1 20.8 ″ 19(K/R-VII) C5(L) 20.2 17.8 ″ 22(K-IX)C4(P) 17.0 18.5 ″ 10(R) C6(R) 16.8 12.9 ″ 25(R-PT) C6(R) 11.2 13.8 ″24(X/D-PC) O(S) 8.1 12.0 ″ 11(K-PT, hX, bS; Gla-PZ) ε-C(K) 14.7 7.43-10-fold^(b) 33(Gla) γ-C(E) 11.6 7.5 ″ 34(D) O(S) 15.3 12.1 ″ 29(R)para-C(F) 7.5 8.4 ″ 16(R) C6(R) 14.2 10.6 3-10-fold^(c) Gla residue^(d)Low importance:  7 12.8 13.3 +2 (<2) 15 20 16 <2 (<2) 20 19.4 17.8 <2(<2) 21 17.2 15 4 (3) 33 11.6 7.5 ?^(e) (<2) High importance:  8 8.710.9 ?^(e) (20) 26 3.6 9.5 ?^(e) (50) 17 11.1 9.1 >200 (100) 27 8.410.6 >200 (85) 30 3.4 4.2 >200 (25)^(a)Distances are from this atom of bovine prothrombin (residue ofprothrombin used in measurement is given in parentheses) to strontium 1and 8 of the Sr-Prothrombin fragment 1 structure. Seshadri et al. 1994,Biochemistry 33: 1087-1092.^(b)For all but 16-R, cations lower affinity and anions increaseaffinity.^(c)Thariath et al. 1997 Biochem. J. 322: 309-315.^(d)Impact of Glu to Asp mutations, distances are averages for thegamma-carboxyl-carbons. K_(D)(K_(M)) data are from Ratcliffe et al. 1993J. Biol. Chem. 268: 24339-45.^(e)Binding was of lower capacity or caused aggregation, makingcomparisons less certain.

The results in FIG. 15C show that the association rate for protein Z wassubstantially improved at pH9, where an amino terminal should beuncharged. The rate constant obtained from these data was about 12-foldhigher at pH9 than at pH7.5 (FIG. 15C).

Example 6 Competition Assay for Assessing Affinity of Modified FactorVII

Clotting activity of wild type and VIIaQ11E33 was assessed usingreconstituted tissue factor (Innovin, Dade) and membrane. Saturatingamounts of factor VIIa were used (approximately 0.7 μl of Innovin/0.15ml assay). Factor VIIa and Innovin were added to the plasma-free buffer(6.7 mM CaCl₂, 50 mM Tris, pH7.5, 100 mM NaCl) in 112.5 μl. After 15minutes, 37.5 μl of factor VII-deficient plasma was added and clottingtime was recorded. Tissue factor-dependent activity of VIIaQ11E33 wassimilar to that of wild-type protein when assayed in this manner. As useof Factor VII-deficient plasma is not representative of in vivoconditions, the relative affinity of modified factor VII formembrane-bound tissue factor was evaluated in the presence of acompeting protein. In particular, active site-modified factor VIIa wasused as the competing protein and was present in abundance (2 nM). Themodified factor VII to be assessed was used above the concentration oftissue factor. Under such conditions, free protein concentrations of allproteins were approximately equal to total proteins added. Subtractionof bound protein from total to obtain free protein concentrationsrepresents a small correction. Based on the competition assay, factorVIIaQ11E33 was 41-times more effective than wild type VIIa.

Example 7 Enhanced Membrane Binding Affinity and Activity of Protein Sand other Vitamin K-Dependent Polypeptides

Protein S (GenBank Accession number M57853 J02917) is a high affinitymembrane-binding protein and a cofactor for action of APC. Deficiency inprotein S is a strong indicator of thrombosis disease and may be used inpatients who have low levels of this protein or who have increaseddanger of thrombosis. See, for example, Dahlback, Blood, 1995,85:607-614 and U.S. Pat. No. 5,258,288.

Substitutions at amino acids 5 or 9 can enhance membrane bindingaffinity and activity of Protein S. Residue 9 of Protein S is athreonine residue, while most vitamin K-dependent proteins contain ahydrophobic residue at this position. See, for example, McDonald et al.,Biochemistry, 1997, 36:5120-5127. Replacement of the analogous residuein human Protein C (a leucine residue) with a glutamine, a hydrophobicto hydrophilic replacement, resulted in a severe loss of activity. See,Christiansen et al., 1995, Biochemistry, 34:10376-10382. Thus, there hasbeen confusion about the importance of position 8 in membraneassociation by vitamin K-dependent proteins.

Human factor VII containing a threonine substitution at amino acid 9 inconjunction with the Q11E33 mutations described above, were prepared byATG Laboratories, Inc. and provided in an appropriate vector fortransfection into human kidney cell line 293, as described above inExample 4.

The vector containing the nucleic acid sequence encoding the mutantfactor VII was transfected into human kidney cell line 293, usingcommercially available kits. The cells were grown and colonies providinghigh levels of factor VII antigen were selected by dot blot assay, asdescribed for protein C, using commercially available polyclonalantibodies. The amount of factor VII in the conditioned medium also wasdetermined by a coagulation assay, using the competition assay describedin Example 6. In general, Factor VII was converted to VIIa by incubationwith tissue factor before initiation of coagulation. Identical amountsof VIIa-Q11E33 and VIIa-T9Q11E33 were used in the assays. The factor VIIpolypeptides were mixed with an appropriate amount ofmembrane-associated tissue factor (Innovin, Dade). Active site modifiedVIIa (FFR-VIIa) was added (0 to 3.5 nM) and the reactions were allowedto equilibrate for 60 minutes in 112.5 μL of buffer containing 6.7 mMCaCl₂, 50 mM Tris, pH7.5, and 100 m NaCl. Finally, factor VII-deficientplasma was added and time required to form a clot was measured. Efficacywas determined by the clotting time as a function of added inhibitor.Human factor VIIa-T9Q11E33 exhibited a severe loss of competitivebinding affinity. Thus, a threonine residue was not optimal at thisposition. Introduction of a leucine residue into protein 9 of protein Sshould enhance the membrane affinity and activity of protein S undermany conditions.

The basis for high membrane affinity of protein S, despite the presenceof substantial sub-optimum residues, may come from other parts of itsstructure. That is, protein S contains a sequence region known as the‘second disulfide loop’ or thrombin sensitive region (residues 46 to 75of the mature polypeptide). Prothrombin also contains a second disulfideloop in a shorter version. Proteolytic cleavage of the loop inprothrombin results in loss of membrane affinity. See, Schwalbe et al.,J. Biol. Chem., 1989, 264:20288-20296. The cleavage may be involved inregulation of protein S activity by providing a negative control toprotein S action. The second disulfide loop may serve to produce anoptimum membrane binding site such that residues 46-75 fold back ontoresidues 1-45 to create an optimum binding site. Isolated residues 1-45of protein S do not associate with membranes in a calcium-dependentmanner, which is unlike residues 1-41 or 1-38 of prothrombin, residues1-44 of factor X, residues 1-41 of protein C, or residues 1-45 ofprotein Z that do bind membranes in a calcium-dependent manner. Thus,despite intact protein S having high affinity, results with the isolated1-45 GLA domain of protein S suggest that the intact protein suffers asubstantial loss of affinity in the GLA domain.

Proteolysis prevents proper function of the second disulfide loop inthis role, and the resultant protein S shows loss of activity as themembrane affinity declines to that expected by the amino acids inresidues 145. Thus, enhancement of membrane affinity by introduction ofLeu9 and other changes (see below), would create a protein S which is nolonger down-regulated by proteolysis and which will be a more effectiveanticoagulant.

A conserved residue in most vitamin K-dependent proteins is position 5.Leucine is found at this position in both protein S and protein Z.Protein C, factors X and VII, and prothrombin contain phenylalanine,another hydrophobic residue, at the corresponding position. Factor IXcontains a lysine at residue 5, a major deviation from other proteins.There appears to be no apparent connection to this unconserved residueand the membrane affinity of factor IX, making the role of position 5ambiguous.

Substitution of a glutamine residue at position 5 of human protein Cresulted in a protein displaying similar affinity for membranes andsimilar calcium titrations, with reduced clotting activity. Substitutionof a leucine for a phenylalanine residue at position 5 of factor VII(which contained the Q11E33 mutations) had much lower activity than theQ11E33 mutant, when assayed by the method of competition withactive-site modified VIIa (0-3.2 nM) described above. Thus, activity ofprotein S can be improved by substitution, for example, of aphenylalanine residue at position 5. The P11 Q mutation of Factor VIIhad a positive impact, while the P11E mutant (to make this position likethat of protein Z), was without detectable impact. Consequently, theimpact of substitutions of individual residues varies with the protein.Appropriate combined substitutions, however, elucidate the universalimportance of these residues.

A factor VIIa molecule was produced by procedures outlined above. Thismutant contained the Q11E33 mutations and also contained R29F and D34Fmutations (Q11F29E33F34 total description). This mutant had 2.5-foldhigher activity than the Q11E33 mutant alone when assayed in thecompetition assay described in Example 6. Thus, the correct combinationof amino acid residues at the important sites described, are needed tomaximize the function of the important carboxyl groups in the protein.The optimum combination at these sites may include anionic as well asneutral and hydrophobic residues.

Example 8 Insertion of Residue at Amino Acid 4

Factor VII containing a tyrosine residue was prepared by ATGLaboratories, Inc. and assayed by the competition assay described inExample 6. Factor VII was activated by incubation with Innovin (20 ml)in 5 mM calcium. Samples containing sufficient amounts of Innovin togive a minimum clotting time of 28 seconds (about 0.7 μl) weretransferred to buffer. Factor VII deficient plasma was added andclotting times were recorded. Samples were assayed at various timesuntil maximum activity was reached (usually ≦30 min.). The amount ofFactor VII in the conditioned media needed to generate a clotting timeof 30 seconds was determined. This ratio of media/Innovin represents anearly 1:1 ratio of factor VIIa/Tissue Factor. After activation, samplesof the media/Innovin sufficient to give a 19 second clotting time (about4 μl of Innovin) were diluted to 112.5 μl with buffer containing calciumand BSA (1 g/L). Various amounts of active-site modified factor VIIa(DEGR-VIIa) were added and allowed to incubate until equilibrium wasreached; typically 60 minutes at 37° C. Human factor VII deficientplasma was added (37.5 μl) and clotting times were measured. Resultswere compared to similar experiments conducted with media containingwild-type VIIa. Based on results from the competitive displacementassay, human factor VII containing a tyrosine residue at position 4 hadtwo-fold higher activity than a similar factor VII molecule lacking thisresidue.

Example 9 Anticoagulation of Human Blood by Activated Protein C (APC)

The Clot Signature Analyzer (CSA) apparatus (Xylum Company, Scarsdale,N.Y.) was used to test relative efficacy of wild type APC versus theQ11G12E32D33 mutant. In one assay, this apparatus pumps freshly drawn(<2 min), non-anticoagulated blood through a tube containing a fiberthat has a collagen surface. The pressure of blood flow (mm Hg) issimilar to that of the circulation system. Platelets in the blood bindto the collagen, become activated and support coagulation. Theinstrument detects pressure at the outlet of the tube, as shown in FIG.16. Upon clot formation, pressure at the outlet of the tube declines andthe half-reaction point is described as the “Collagen-Induced ThrombusFormation” (CITF) time. Without additives, the CITF time for the humansubject was 5.0 minutes (FIG. 16A). Background time is subtracted fromthe value in FIG. 16 to obtain CITF. With 30 nM wild type APC added tothe blood, the average CITF time was 6.5 min (5 determinations) for thesame subject (FIG. 16B). The average CITF time was 15.5 min with 6 nMmutant APC (Q11G12E32D33), based on 5 determinations (FIG. 16C), and was9.5 min with 3 nM of the same mutant APC, based on 5 determinations.Thus, the mutant APC was at least 10-fold more effective than wild typeAPC for inhibition of clot formation in whole human blood under flowpressure, using activated human platelet membranes as the phospholipidsource. In one additional experiment, in which CITF was assessed in asubject that had ingested two aspirins, an even larger difference wasobserved between mutant and wild type APC. Lower membrane activity maycorrelate with a larger impact of the mutant.

This result differed from the outcome of an in vitro coagulation testusing the hand tilt assay procedure, described earlier in this document.Using the standard conditions and full strength reagents for the APTTtest (materials obtained from, and procedures described by themanufacturer, the Sigma Chemical Co., St. Louis, Mo., 1998), the mutantprotein showed only 1.5-fold higher activity than the wild type protein.The APTT assay, however, contains high levels of phospholipid, acondition that minimizes the difference between mutant and wild typeproteins. Once again, these results indicate that assay conditions areimportant in characterizing the benefit of the mutant proteins producedby this invention and that low phospholipid concentration ischaracteristic of the biological membrane provided by platelets.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-60. (canceled)
 61. A composition comprising an anticoagulant agent anda protein C or activated protein C polypeptide comprising a modified GLAdomain, said modified GLA domain comprising the amino acid sequence ofSEQ ID NO:1 with one, two, three, four, or five amino acid substitutionsat positions selected from the group consisting of residues 10, 11, 28,32, and
 33. 62. The composition of claim 61, wherein said anticoagulantagent is aspirin, warfarin, or heparin.
 63. The composition of claim 61,wherein said anticoagulant agent is aspirin.
 64. The composition ofclaim 61, wherein said one amino acid substitution is at residue
 10. 65.The composition of claim 61, wherein said one amino acid substitution isat residue
 11. 66. The composition of claim 61, wherein said one aminoacid substitution is at residue
 28. 67. The composition of claim 61,wherein said one amino acid substitution is at residue
 33. 68. Thecomposition of claim 61, wherein said modified GLA domain comprises theamino acid sequence of SEQ ID NO:1 with three amino acid substitutionsat residues 11, 32, and
 33. 69. The composition of claim 68, whereinresidue 32 of SEQ ID NO:1 is glutamic acid and residue 33 of SEQ ID NO:1is aspartic acid.
 70. The composition of claim 69, wherein residue 11 ofSEQ ID NO:1 is glycine.